Continuous isolation of cannabidiol and conversion of cannabidiol to delta 8-tetrahydrocannabinol and delta 9-tetrahydrocannabinol

ABSTRACT

In alternative embodiments, provided are processes comprising the continuous isolation and purification of cannabinoids and further isomerization of the purified cannabidiol to Δ8tetrahydrocannabinol (Δ8THC) and Δ9tetrahydrocannabinol (Δ9THC). In alternative embodiments, provided are processes for converting Δ8-THC into Δ9-THC. In alternative embodiments, provided are processes for the industrial scale continuous isolation and purification of cannabinoids and further isomerization of the purified cannabidiol to Δ9-THC.

This application is a divisional patent application under 35 U.S.C. § 120 claiming benefit of priority to U.S. patent application Ser. No. 16/388,182, filed Apr. 18, 2019 (now pending), which continuation of Patent Convention Treaty (PCT) International Application serial number PCT/US2019/021138, filed Mar. 7, 2019, which claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. (USSN) U.S. Ser. No. 62/639,608, filed Mar. 7, 2018; U.S. Ser. No. 62/697,920, filed Jul. 13, 2018; U.S. Ser. No. 62/697,926, filed Jul. 13, 2018; U.S. Ser. No. 62/697,923, filed Jul. 13, 2018; and U.S. Ser. No. 62/715,545, filed Aug. 7, 2018. The aforementioned applications are expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

FIELD OF THE INVENTION

This invention generally relates to the industrial scale processing and purification of cannabinoids. In alternative embodiments, provided are processes comprising the continuous isolation and purification of cannabinoids and further isomerization of the purified cannabidiol to Δ⁸-tetrahydrocannabinol (Δ⁸-THC) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC). In alternative embodiments, provided are processes for converting Δ⁸-THC into Δ⁹-THC. In alternative embodiments, provided are processes for the industrial scale continuous isolation and purification of cannabinoids and further isomerization of the purified cannabidiol to Δ⁹-THC.

BACKGROUND

Cannabinoids are a class of diverse chemical compounds that acts on cannabinoid receptors in cells that alter neurotransmitter release in the brain. They are of great value in pharmaceutical applications. Ligands for these receptor proteins include the endocannabinoids (produced naturally in the body by animals) and the phytocannabinoids (found in hemp, Cannabis and some other plants). Among these cannanbinoids is cannabidiol (CBD) widely considered to have a range of potential medical applications including treatment of Dravet syndrome, a rare form of epilepsy that is difficult to treat that begins in infancy. Another cannabinoid is Δ⁹tetrahydrocannabinol (Δ⁹THC) that acts as an appetite stimulant for people with AIDS and antiemetic for people receiving chemotherapy.

Tetrahydrocannabinol (THC) is the principal psychoactive constituent of Cannabis. Natural phytocannabinoids sources such as hemp and Cannabis contain at least 113 different cannabinoids. Currently mixtures comprising terpenes, lipids, waxes, chlorophyll and other plant matter, including cannabinoids, are extracted in mass using polar solvent extraction, nonpolar solvent extraction or mixtures thereof and then physically separated using multiple steps including distillation, chromatography, winterization or combinations of these processes.

Recently, public interest in Cannabis as medicine has been growing, based in no small part on the fact that Cannabis has long been considered to have medicinal properties, ranging from treatment of cramps, migraines, convulsions, appetite stimulation and attenuation of nausea and vomiting. In fact, a report issued by the National Academy of Sciences' Institute of Medicine indicated that the active components of Cannabis appear to be useful in treating pain, nausea, AIDS-related weight loss or “wasting”, muscle spasms in multiple sclerosis as well as other problems. Advocates of medical marijuana argue that Cannabis is also useful for glaucoma, Parkinson's disease, Huntington's disease, migraines, epilepsy and Alzheimer's disease.

Marijuana refers to varieties of Cannabis having a high content of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), which is the psychoactive ingredient of marijuana, whereas industrial hemp refers to varieties of the Cannabis plant that have a low content of Δ⁹-THC.

Furthermore, Δ⁹-THC is only one of a family of about 60 bi- and tri-cyclic compounds named cannabinoids. For example, Δ⁸-THC is a double bond isomer of Δ⁹-THC and is a minor constituent of most varieties of Cannabis (see, e.g., Hollister and Gillespie, 1972, Clin Pharmacol Ther 14: 353). The major chemical difference between the two compounds is that Δ⁹-THC can be oxidized to cannabinol whereas Δ⁸-THC does not and is in fact very stable. Δ⁸-THC, for the most part, produces similar psychometric effects as does Δ⁹-THC, but is generally considered to be 50% less potent than Δ⁹-THC and has been shown in some cases to be 3-10 times less potent. Δ⁸-THC has also been shown to be more (200%) effective an anti-emetic than Δ⁹-THC and has been used as an anti-emetic in children, based on the belief that the side effects of Δ⁹-THC and Δ⁸-THC, such as anxiety and dysphoria, are more prevalent in adults than children (Abrahamov et al, 1995, Life Sciences 56: 2097-2102). On the other hand, CBD has no activity on its own when administered to humans. It is of note that CBD is typically about 2% (0.54%) dry weight of hemp chaff, Δ⁸-THC is approximately 0.2% (0.05-0.5%) dry weight and Δ⁹-THC is approximately 0.1% (0.05-0.3%).

The large variety of cannabinoids and other plant matter present in extracts makes it difficult to separate the cannabinoids from each other as well as from the non-cannabinoid plant matter and achieve a high enough purity level to meet pharmaceutical or analytical requirements. Separations can be achieved using various techniques including such as distillation which subjects the cannabinoid rich mixture of heat history which can degrade the cannabinoids. For separations that do not subject the cannabinoids to heat history chromatography is applied however the current state of the art is to apply these techniques in a non-continuous method which severely limits the scale and efficiency of the separation methodology.

There remains a need for a method that is simple, efficient and continuous process to first generate purified cannabidiol and combined with a simple, efficient method to convert cannabidiol into a mixture of Δ⁸THC and Δ⁹-THC. Clearly, as the cannabinoids are of potential medicinal value, improved methods of both isolating CBD and converting CBD to Δ⁸-THC and Δ⁹-THC are needed.

SUMMARY

Chapt. I

In alternative embodiments, provided are processes and methods for purifying cannabinoids and converting cannabidiol to Δ⁸-tetrahydrocannabinol (Δ⁸THC) and Δ⁹-tetrahydrocannabinol (Δ⁹THC). In alternative embodiments, provided are processes for converting Δ⁸-THC to Δ⁹-THC.

In alternative embodiments, provided are processes and methods for obtaining or purifying a substantially pure Δ⁹ THC from a natural or a synthetic source, wherein optionally the natural source comprises a plant or a microbial material, or a material derived from a plant source, comprising:

-   -   (a) obtaining or being provided a first extract, aliquot or         sample of the natural or synthetic source comprising a         cannabidiol (CBD) and/or Δ⁹ THC and/or Δ⁸ THC and/or other         cannabinoids from the natural or the synthetic source;     -   (b) dissolving the first extract aliquot or sample in a first         solvent, or diluting the first extract aliquot or sample in a         first solvent;     -   (c) loading the solvent-dissolved or diluted first extract         aliquot or sample onto a first chromatography column, wherein         optionally the chromatography column is a normal phase         chromatography column, an ion exchange chromatography column, or         a reverse phase chromatography column,     -   wherein the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids are         reversibly bound to the first chromatography column,     -   wherein optionally some of or substantially most of the extract         aliquot or sample material not reversibly bound to the first         column at the first station is removed with a first wash         solution before the column is moved to a next or a second         station;     -   (d) eluting the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids         off the first chromatography column with an elution solvent         mixture, wherein optionally the eluting is a gradient elution;     -   (e) collecting or isolating the Δ⁹ THC and/or Δ⁸ THC and/or         other cannabinoids from said elution solvent mixture,     -   wherein optionally the elution is by a gradient elution process         and the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids are         separately collected in different or separate gradient elution         fractions,     -   wherein optionally, if the elution is by a gradient elution         process using normal phase column chromatography: in a first         gradient polar compounds and some or substantially most of the         cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC) are         eluted off the column; in a second gradient solvent remaining         cannabidiol (CBD) is eluted off the column; in a third gradient         a CBD/tetrahydrocannabinol (THC) mix is first eluted and then         remaining THC is eluted off the column; in a fourth gradient         remaining cannabinoids are eluted off the column, wherein         optionally the remaining cannabinoids comprise CBC, CBG, CBN;         and, in a fifth gradient solvent all remaining polar compounds         are eluted off the column, wherein the remaining polar compounds         comprises cannabidiolic acid (CBDA) and tetrahydrocannabinolic         acid (THCA),     -   and optionally, if the elution is by a gradient elution process         using reverse phase column chromatography, the gradient elution:         -   begins with an 80:20 or a 70:30 methanol to water elution             solution or equivalent, which elutes off the column some or             substantially most of the polar compounds, wherein             optionally the polar compounds comprise CBDA and/or TGCA;         -   then the gradient elution moves to an 85:15 or an 80:20             methanol to water elution solvent or equivalent, which             elutes off the column some or substantially most of the CBD;         -   then the gradient elution moves to a 90:10 methanol to water             elution solution or equivalent, which initially elutes off             the column a mix of the remaining CBD and THC, and finally             elutes off the column THC; and         -   finally the gradient elution moves to a 100% methanol             elution solution, which removes a mix of remaining             cannabinoids comprising CBC, CBG, CBN;     -   (f) adding a second solvent of opposite polarity (to the elution         solvent mixture) to said elution solvent mixture;     -   (g) removing all or substantially most of the second solvent,         thereby leaving a first extract comprising a plurality of         cannabinoids comprising the Δ⁹ THC and/or Δ⁸ THC and/or other         cannabinoids, wherein optionally the other cannabinoids comprise         CBC, CBG, CBN;     -   (h) dissolving the first extract comprising the plurality of         cannabinoids in a first reaction solvent;     -   (i) adding a catalyst to the first extract comprising the         plurality of cannabinoids and the first reaction solvent,         wherein the catalyst can be added before, simultaneous with or         after addition of the first reaction solvent to the extract         comprising the plurality of cannabinoids; and     -   (j) reacting the first extract comprising the plurality of         cannabinoids, the catalyst and the first reaction solvent,         wherein optionally the reaction converts Δ8-THC to Δ⁹-THC;     -   (k) adding a neutralizing agent;     -   (l) removal of the catalyst and the neutralizing agent to         generate a first reaction product;     -   (m) optionally adding a second reaction solvent to the first         reaction product;     -   (n) optionally removing all or substantially most of the second         reaction solvent, thereby leaving a second extract comprising a         plurality of cannabinoids;     -   (o) adding a stabilization agent to the second extract;     -   (p) adding an elimination agent to the second extract;     -   (q) removal (or removal of substantially all) of the         stabilization agent and the elimination agent, thereby leaving a         third extract;     -   (r) and if the second reaction solvent is not removed in step         (n), adding a third solvent of opposite polarity to the third         extract;     -   (s) removing the second reaction solvent, if present (if not         removed in step (n)), and removing all or substantially most of         the third solvent of opposite polarity, if added, thereby         leaving a fourth extract;     -   (t) dissolving third or the fourth extract in a second solvent,         wherein optionally the second solvent is the same or         substantially the same as the first solvent;     -   (u) loading the dissolved third or the fourth extract onto a         second chromatography column, wherein optionally the         chromatography column is a normal phase chromatography column or         a reverse phase chromatography column,     -   wherein the plurality of cannabinoids are reversible bound to         the second chromatography column, and optionally the second         chromatography column is the same as the first chromatography         column;     -   (v) eluting the dissolved third or the fourth extract from the         second column with an elution solution,     -   wherein optionally the elution is by a gradient elution process         and the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids are         separately collected in different or separate gradient elution         fractions;     -   (w) optionally collecting the plurality of cannabinoids from the         elution solution, and/or removing all or substantially most of         the elution solvent,     -   thereby obtaining or purifying a substantially pure plurality of         cannabinoids, wherein optionally the plurality of cannabinoids         comprises Δ⁹ THC and/or Δ⁸ THC.

In alternative embodiments, the CBD content of the first extract, aliquot or sample material is greater than about 25%, 50%, 75%, 90% or 95% by weight, or is between about 5% and 95% by weight, by weight of the material.

In alternative embodiments, the first solvent and/or the second solvent comprises methanol, propanol, ethanol, isobutanol, butanol, isopropanol, tetrahydrofuran, chloroform, acetone, hexane, methylene dichloride, and/or dichloroethane; and optionally the first solvent and/or the second solvent comprises chloroform, methylene dichloride and methanol, propanol and/or ethanol.

In alternative embodiments, the chromatography column, e.g., a normal phase chromatography column, ion exchange column or reverse phase column, comprises a silica gel, an alumina or an ion exchange material, and optionally the ion exchange column comprises an anion exchange material.

In alternative embodiments, the elution solution comprises or is a solvent, and the solvent comprises a one or a mixture of: ethylene dichloride; chloroform; or methylene dichloride; and alcohol comprising methanol, ethanol, propanol or butanol or a mixture thereof; an acid comprising acetic acid, formic acid, oxalic acid, glycolic acid or a mixture thereof; and/or, ammonium hydroxide. In alternative embodiments, the elution solvent is or comprises a dichloromethane, dichloroethane, or chloroform and methanol or ethanol.

In alternative embodiments, the first reaction solvent comprises ethylene dichloride, chloroform, methylene dichloride, toluene, xylene, benzene, hexane and/or pentane, or mixtures thereof.

In alternative embodiments, the catalyst is or comprises a Lewis acid or a non-oxidizing acid catalyst. In alternative embodiments, the catalyst comprises p-toluenesulfonic acid.

In alternative embodiments, the neutralizing agent is or comprises an alkali. In alternative embodiments, the neutralizing agent is or comprises sodium bicarbonate, sodium carbonate, calcium hydroxide or ammonium hydroxide.

In alternative embodiments, the stabilization agent is or comprises zinc chloride or methylene chloride.

In alternative embodiments, the second reaction solvent comprises dimethyl sulfoxide, water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, benzene, toluene and/or xylene or a mixture thereof. In alternative embodiments, the second reaction solvent comprises chloroform, hexane, dichloromethane, dichloroethane, benzene or toluene or a mixture thereof.

In alternative embodiments, the elimination agent is or comprises a strong base; and optionally the strong base comprises: lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)₂), calcium hydroxide (Ca(OH)₂), strontium hydroxide Sr(OH)₂), barium hydroxide Ba(OH)₂), potassium hydride and/or sodium hydride, potassium tert-pentoxide, organic superbases, bispidines, multicyclic polyamines, organometallic compounds of reactive metals, wherein optionally the reactive metals comprise organolithium, organo-magnesium, lithium diisopropylamide, n-butyl lithium and potassium tert-butoxide, sodium methoxide, or sodium ethoxide. In alternative embodiments, the elimination agent is or comprises potassium tert-pentoxide, lithium diisopropylamide and/or potassium tert-butoxide.

In alternative embodiments, the elution solvent is or comprises a solvent or mixture of solvents, e.g., as described in the Examples.

In alternative embodiments, the extract is filtered before being loaded on to the column, and optionally the extract is mixed with silica, diatomaceous earth (DE), bentonite clay (BC), or equivalents, or mixtures thereof, before filtering, and optionally the extract is chilled before being mixed with the silica, DE, BC or equivalents or is chilled before being filtered.

In alternative embodiments, the microbial material comprises or is derived from a bacterium, an algal cell, a lichen or a yeast, or a recombinant bacterium, lichen, algal cell or yeast cell.

In alternative embodiments, the plant or starting material comprises or is derived from a botanical drug substance, including hemp or a Cannabis plant, wherein optionally the Cannabis plant is a Cannabis sativa, a Cannabis chemovar, or variants thereof, or a decarboxylated Cannabis plant material. In alternative embodiments, the plant or starting material comprises oils or extracts from a trichome or a trichome fraction of a pubescent plant, or an algae or a lichen; and optionally the oils or extracts are from the plant or microbial material by: washing, contacting or exposing the trichome or trichome fraction, or the pubescent plant, algae or lichen, with: at least one non-polar, organic solvent; at least one polar, organic solvent; or, a mix of at least one non-polar, organic solvent with (and) at least one polar, organic solvent.

Chapt. II

In alternative embodiments, provided are methods of or processes for purifying a cannabinoid to substantially pure Δ⁹tetrahydrocannabinol (Δ⁹-THC) from a natural or a synthetic source, wherein optionally the natural source comprises a plant or microbial material, or a material derived from a plant or a microbial source.

In one embodiment, the method comprises:

-   -   a. providing or having provided a first extract, aliquot or         sample of the natural or synthetic source comprising at least         one of cannabidiol (CBD), Δ⁹ THC, Δ⁸ THC, and other cannabinoids         from the natural or the synthetic source, optionally a plant         material;     -   b. providing or having provided a continuous chromatography         apparatus or device comprising a plurality of stations and a         plurality of chromatography columns, wherein optionally the         plurality of stations and the plurality of chromatography         columns comprise at least 2, 3, 4, 5, 6, 7, or 8 or more, or         between about 3 and 30, stations and/or chromatography columns;     -   c. introducing or loading the first extract, aliquot or sample         into a first column at a first station, wherein the CBD, Δ⁹ THC,         and Δ⁸ THC and other cannabinoids reversibly bind to the first         column,     -   wherein optionally some of or substantially most of the first         extract material not reversibly bound to the first column at the         first station is removed with a first wash solution before the         column is moved to a next or a second station;     -   d. moving the first column to a second station, wherein after         the first column is moved to the second station a next or second         column is introduced into the first station;     -   e. eluting the extract from the first chromatography column with         a first elution solvent at the second station, wherein         optionally the eluting is a gradient elution;     -   f. collecting the first elution fractions from the first column         at the second station;     -   g. removing all or substantially most of the first elution         solution from the first elution fractions to produce a first         purified extract substantially free of CBD, Δ⁹ THC, and Δ⁸ THC;     -   h. optionally repeating steps d to g on the first column with at         least one additional elution solvent, wherein the at least one         additional elution solvent produces a second purified extract         substantially free of CBD, Δ⁹ THC, and Δ⁸ THC;     -   i. when the first column is moved to the next, or second,         station, introducing a next or second column into the first         station, and the extract, aliquot or sample is introduced or         loading into the next or second column at the first station;     -   j. moving the second column to the next or second station after         the first column is moved to a next or third station;     -   k. eluting the extract from the first column with a second         elution solvent at the next or second station,     -   wherein optionally the elution is by a gradient elution process         and the CBD, Δ⁹ THC, and Δ⁸ THC and other cannabinoids are         separately collected in different or separate gradient elution         fractions     -   wherein optionally, if the elution is by a gradient elution         process: in a first gradient solvent non-polar compounds and         terpenes are eluted off the column; in a second gradient solvent         cannabidiol (CBD) is eluted off the column; in a third gradient         solvent a CBD/tetrahydrocannabinol (THC) mix is first eluted and         then remaining THC is eluted off the column; in a fourth         gradient minor cannabinoids are eluted off the column, wherein         optionally the minor cannabinoids comprise CBC, CBG, CBN; and,         in a fifth gradient solvent all remaining polar compounds are         eluted off the column, wherein the remaining polar compounds         comprises cannabidiolic acid (CBDA) and tetrahydrocannabinolic         acid (THCA);     -   l. collecting the second elution fractions from the first         chromatography column at the second station;     -   m. removing all or substantially most of the second elution         solvent from the second elution fractions to produce a second         purified extract (or a third purified extract if step h is         completed) comprising at least about 75% of the CBD, Δ⁹ THC, and         Δ⁸ THC from the first extract, or between about 60% and 90% of         CBD, Δ⁹ THC, and Δ⁸ THC, or between about 50% and 95% of CBD, Δ⁹         THC, and Δ⁸ THC,     -   and optionally the gradient elution:         -   begins with an 80:20 or a 70:30 methanol to water elution             solution or equivalent, which elutes off the column some or             substantially most of the cannabidiolic acid (CBDA) and             tetrahydrocannabinolic acid (THCA) and polar terpenes;         -   then the gradient elution moves to an 85:15 an 80:20             methanol to water elution solvent or equivalent, which             elutes off the column some or substantially most of the CBD;         -   then the gradient elution moves to a 90:10 methanol to water             elution solution or equivalent, which initially elutes off             the column a mix of the remaining CBD and THC, and finally             elutes off the column THC; and         -   finally the gradient elution moves to a 100% methanol             elution solution, which removes a mix of remaining             cannabinoids comprising CBC, CBG, CBN;     -   n. optionally repeating steps j to m on the first column with at         least one second additional elution solution;     -   o. optionally repeating steps i to m with at least one         additional column;     -   p. optionally mixing the second purified extract with a reaction         mixture comprising a reaction solvent and a catalyst, wherein         the reaction mixture comprises an aqueous phase and an organic         phase;     -   q. optionally separating the mixture into an aqueous phase and         an organic phase (separating the aqueous phase and the organic         phase): and     -   r. optionally recovering the organic phase to obtain the         substantially pure Δ⁹-THC.

In some embodiments, the method further comprises: adding a neutralizing agent to the reaction mixture; and removing all or substantially most of the catalyst and the neutralizing agent before separating the reaction mixture.

In some embodiments, the method further comprises: adding at least one of a stabilization agent and an elimination agent to the reaction mixture; and removing all or substantially most of the at least one of the stabilization agents and the elimination agent before separating the reaction mixture.

In some embodiments, the extract, aliquot or sample comprises at least 25% by weight CBD, or between about 10% to 40% by weight CBD, or between about 5% to 50% by weight CBD.

In some embodiments, at least one of the first elution solvent, the additional elution solvent, the second elution solvent, and the second additional elution solvent comprises methanol, ethanol, propanol, butanol, chloroform, dichloromethane, dichloroethane, acetic acid, formic acid, oxalic acid, glycolic acid, ammonium hydroxide, or combinations thereof.

In some embodiments, the reaction solvent comprises ethylene dichloride, chloroform, methylene dichloride, toluene, xylene, benzene, hexane, pentane, or combinations thereof.

In some embodiments, the catalyst comprises a Lewis acid or a non-oxidizing acid. In some embodiments, the catalyst comprises p-toluenesulfonic acid

In some embodiments, the neutralizing agent comprises an alkali. In some embodiments, the neutralizing agent comprises sodium bicarbonate, sodium carbonate, calcium hydroxide, or ammonium hydroxide, or combinations thereof.

In some embodiments, the stabilization agent is or comprises zinc chloride or methylene chloride, or combinations thereof.

In some embodiments, the method further comprises: adding a second reaction solvent to the organic phase after the mixture is separated.

In some embodiments, the second reaction solvent comprises dimethyl sulfoxide, pyridine, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, benzene, toluene, xylene, or combinations thereof.

In some embodiments, the elimination agent comprises lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)₂), calcium hydroxide (Ca(OH)₂), strontium hydroxide Sr(OH)₂), barium hydroxide Ba(OH)₂), potassium hydride and/or sodium hydride, potassium tert-pentoxide, organic superbases, bispidines, multicyclic polyamines, or organometallic compounds of reactive metals, wherein optionally the reactive metals comprise organolithium, organomagnesium, lithium diisopropylamide, n-butyl lithium and potassium tert-butoxide, sodium methoxide, or sodium ethoxide, lithium nitride, potassium hydride, sodium hydride, or combinations thereof.

In some embodiments, the method further comprises: dissolving, filtering and/or diluting the extract, aliquot or sample in a first solvent before introducing or loading the extract, aliquot or sample into the first chromatography column, or the column at the first station, which can be a second, third, fourth etc. chromatography column.

In some embodiments, the first solvent comprises methanol, ethanol, propanol, isobutanol, butanol, isopropanol, tetrahydrofuran, chloroform, methylene dichloride, dichloroethane, or combinations thereof.

In some embodiments, the method further comprises: filtering the extract, sample or aliquot to remove all or substantially most of the solids and color bodies in the natural or synthetic material before introducing or loading the extract, aliquot or sample into the first column, or any additional column at the first station.

In some embodiments, at least one of the first chromatography column, the second chromatography column, or the at least one additional chromatography column comprises a normal phase chromatography column, a reverse phase chromatography column, or an ion exchange chromatography column.

In some embodiments, the method further comprises measuring the amount of Δ⁹-THC, or other cannabinoid, in the organic phase.

In some embodiments, the first and second elution solutions are different.

In some embodiments, there are at least 4 columns, at least four elution solvents, and at least four purified extracts, or between about 3 and 30 elution solvents and purified extracts. In some embodiments, the at least four elution solvents are different.

In alternative embodiments, methods and processes comprise a continuous column chromatography process for isolating and/or modifying cannabidiol (CBD) and/or Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids, comprising:

-   -   (a) at a first station, loading onto a first column the extract,         aliquot or sample of the natural or synthetic source comprising         a cannabidiol (CBD) and/or Δ⁹ THC and/or Δ⁸ THC and/or other         cannabinoids from the natural or the synthetic source, and then         moving the first column to a second station;     -   (b) at the second station, introducing into the first column an         elution solvent comprising an 80:20 or a 70:30 methanol to water         elution solution or equivalent, which elutes off the first         column some or substantially most of the polar compounds and         terpenes comprising cannabidiolic acid (CBDA) and         tetrahydrocannabinolic acid (THCA), and then moving the first         column to a third station;     -   (c) at the third station, introducing into the first column an         elution solvent comprising an 85:15 or an 80:20 methanol to         water elution solvent or equivalent, which elutes off the first         column some or substantially most of the CBD, and then moving         the first column to a fourth station;     -   (d) at the fourth station, introducing into the first column an         elution solvent comprising a 90:10 methanol to water elution         solution or equivalent, which elutes off the first column the         minor CBD, and some THC, and finally elutes off the column more         or substantially most of the THC, then moving the first column         to a fifth station;     -   (e) at the fifth station, introducing into the first column an         elution solution comprising a 100% methanol elution solution,         which removes a mix of remaining cannabinoids comprising CBC,         CBG, CBN, then moving the first column to a sixth station;     -   (f) at the sixth station, introducing into the first column an         elution solution comprising dichloromethane (DCM), chloroform or         equivalent to remove or elute off the non-polar compounds         reversibly bound to the column, wherein optionally the non-polar         compounds comprise cannabidiol (CBD), cannabinol (CBN),         cannabigerol (CBG), cannabichromene (CBC), cannabidivarol         (CBDV), tetrahydrocannabidiol (THCBD), tetrahydrocannabigerol         (THCBG), tetrahydrocannabichromene (THCBC), or         tetrahydrocannabidivarol (THCBDV), then moving the first column         to a seventh station or back to the first station; and     -   (g) introducing into the first column an 80:20 or a 70:30         methanol to water elution solution or equivalent either at the         seventh station or the first station in an amount sufficient to         remove all or substantially most of the dichloromethane (DCM),         chloroform or equivalent, and if the 80:20 or the 70:30 methanol         to water elution solvent or equivalent is introduced at the         seventh station, after remove all or substantially most of the         dichloromethane (DCM), chloroform or equivalent the first column         is moved back to the first station;     -   wherein one or several fractions are collected at each station,         or at least at the first to fifth stations or first to sixth         stations.

In alternative embodiments, the method or processes further comprise mixing the isolated cannabidiol (CBD) and/or Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoid fractions with a reaction solvent and a catalyst, for example, for converting cannabidiol (CBD) to Δ⁸-THC and/or Δ⁹-THC, or for converting Δ⁸-THC to Δ⁹-THC.

In alternative embodiments, provided are methods of purifying a cannabinoid to substantially pure Δ⁹-THC from a natural or a synthetic source, e.g., a plant or a microbial material, comprising:

-   -   a. providing an extract, aliquot or sample of the natural or         synthetic source comprising at least one of cannabidiol (CBD),         Δ⁹ THC, THC, and other cannabinoids from the natural or a         synthetic source, wherein the natural or a synthetic source         comprises a plant or a microbial material;     -   b. loading the CBD-, Δ⁹ THC-, Δ⁸ THC-comprising extract, aliquot         or sample on a chromatography column,     -   wherein optionally the chromatography column is a normal phase         chromatography column, an ion exchange chromatography column, or         a reverse phase chromatography column, and     -   eluting the column with a first elution solution so that all or         substantially most of the CBD, Δ⁹ THC, Δ⁸ THC remains in (or         reversibly bound to) the column and the all or substantially         most of the remaining natural or synthetic, optionally plant or         microbial, material is eluted out of the column;     -   c. collecting the eluted material and removing all or         substantially most of the first elution solution, wherein the         first elution solution is substantially free of CBD, Δ⁹ THC, Δ⁸         THC;     -   d. eluting some or substantially most of the CBD, Δ⁹ THC and/or         Δ⁸ THC from the column with a second elution solution, wherein         optionally between about 50% and 99% of the CBD, Δ⁹ THC and/or         Δ⁸ THC is eluted from the column, and     -   collecting the CBD, Δ⁹ THC, Δ⁸ THC, or collecting the CBD, Δ⁹         THC, Δ⁸ THC elution fractions, wherein one or multiple fractions         are collected, and optionally the CBD, Δ⁹ THC, Δ⁸ THC are         collected in separate elution fractions, and     -   removing all or substantially most of the second elution         solution,     -   wherein optionally the elution of step (d) is by a gradient         elution process,     -   and optionally the elution is by a gradient elution process and         the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids are         separately collected in different or separate gradient elution         fractions,     -   wherein optionally, if the elution is by a gradient elution         process: in a first gradient solvent non-polar compounds and         terpenes are eluted off the column; in a second gradient solvent         cannabidiol (CBD) is eluted off the column; in a third gradient         a CBD/tetrahydrocannabinol (THC) mix is first eluted and then         remaining THC is eluted off the column; in a fourth gradient         minor cannabinoids are eluted off the column, wherein optionally         the minor cannabinoids comprise CBC, CBG, CBN; and, in a fifth         gradient solvent all remaining polar compounds are eluted off         the column, wherein the remaining polar compounds comprises         cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid         (THCA);     -   e. loading the collected CBD, Δ⁹ THC, Δ⁸ THC on a second         chromatography column and eluting the CBD from the second column         with a third elution solution (the Δ⁹ THC and Δ⁸ THC stay on the         second column), collecting the second eluted material containing         (or comprising) the CBD, and removing all or substantially most         of the third elution solvent;     -   f. eluting the Δ⁹ THC and Δ⁸ THC from the second column with a         fourth elution solution, collecting the fraction, and removing         all or substantially most of the fourth elution solution;     -   g. adding the CBD from the second column to a reaction solvent         and adding a catalyst forming a reaction mixture, wherein the         reaction mixture comprises an aqueous phase and an organic         phase.

In some embodiments, steps b to d are continuously repeated and wherein steps e to g are continuously repeated.

In some embodiments, the method further comprises: adding a neutralizing agent to the mixture; and removing all or substantially most of the catalyst and the neutralizing agent before separating the mixture, and optionally separating the aqueous phase from the organic phase.

In some embodiments, the method further comprises: adding at least one of a stabilization agent and an elimination agent to the mixture; and removing all or substantially most of the at least one of the stabilization agent and the elimination agent before separating the mixture.

In some embodiments, the extract comprises at least 25% CBD by weight, or between about 10% to 40% CBD by weight, or between about 5% to 50% CBD by weight, by weight.

In some embodiments, at least one of the first elution solvent, the second elution solvent, the third elution solvent, and the fourth additional elution solvent comprises methanol, ethanol, propanol, butanol, chloroform, dichloromethane, dichloroethane, acetic acid, formic acid, oxalic acid, glycolic acid, ammonium hydroxide, water, or combinations thereof.

In some embodiments, the reaction solvent comprises ethylene dichloride, chloroform, methylene dichloride, toluene, xylene, benzene, hexane, pentane, water, or combinations thereof.

In some embodiments, the catalyst comprises a Lewis acid or a non-oxidizing acid. In some embodiments, the catalyst comprises p-toluenesulfonic acid

In some embodiments, the neutralizing agent comprises an alkali. In some embodiments, the neutralizing agent comprises sodium bicarbonate, sodium carbonate, calcium hydroxide or ammonium hydroxide.

In some embodiments, the stabilization agent is or comprises zinc chloride or methylene chloride.

In some embodiments, the method further comprises: adding a second reaction solvent to the organic phase after the mixture is separated.

In some embodiments, the second reaction solvent comprises dimethyl sulfoxide, pyridine, water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, benzene, toluene, xylene, or combinations thereof.

In some embodiments, the elimination agent comprises lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)₂), calcium hydroxide (Ca(OH)₂), strontium hydroxide Sr(OH)₂), barium hydroxide Ba(OH)₂), potassium hydride and/or sodium hydride, potassium tert-pentoxide, organic superbases, bispidines, multicyclic polyamines, organometallic compounds of reactive metals, wherein optionally the reactive metals comprise organolithium, organomagnesium, lithium diisopropylamide, sodium methoxide, or sodium ethoxide, or combinations thereof.

In some embodiments, the method further comprises: dissolving, diluting and/or filtering the extract, aliquot or sample comprising the natural or synthetic material in a first solvent before introducing or loading the extract, aliquot or sample into the first column, or before the initial loading of the second or additional columns.

In some embodiments, the first solvent comprises methanol, ethanol, propanol, isobutanol, butanol, isopropanol, tetrahydrofuran, chloroform, methylene dichloride, dichloroethane, water, or combinations thereof.

In some embodiments, the method further comprises: filtering the extract, aliquot or sample comprising the natural or synthetic material to remove all or substantially most of the material's solids and color bodies before introducing or loading the extract, aliquot or sample into the first column, or before the initial loading of the extract, aliquot or sample in the second or additional columns.

In some embodiments, at least one of the columns, or the first and/or the second column comprises a normal phase chromatography column, a reverse phase chromatography column, or an ion exchange chromatography column. In alternative embodiments, all of the columns are the same (e.g., all are normal phase chromatography columns or reverse phase chromatography columns), or the columns differ (e.g., some are normal phase chromatography columns and some are reverse phase chromatography columns or ion exchange chromatography columns).

In some embodiments, the method further comprising measuring the amount of Δ⁹-THC, or other cannabinoid, in the organic phase.

In some embodiments, the first, second, third, and fourth elution solutions are different.

In alternative embodiments, the natural or synthetic material, or the extract, aliquot or sample, is filtered before being loaded on to the column, and optionally the natural or synthetic material, or the extract, aliquot or sample, is mixed with silica, diatomaceous earth (DE), bentonite clay (BC), or equivalents, or mixtures thereof, before filtering, and optionally the natural or synthetic material, or the extract, aliquot or sample, is chilled before being mixed with the silica, DE, BC or equivalents or is chilled before being filtered.

In alternative embodiments, the microbial material comprises or is derived from a bacterium, an algal cell, a lichen or a yeast, or a recombinant bacterium, algal cell, lichen or yeast cell. In alternative embodiments, the plant material comprises or is derived from a botanical drug substance, including a hemp or a Cannabis plant, wherein optionally the Cannabis plant is a Cannabis sativa, a Cannabis chemovar, or variants thereof, or a decarboxylated Cannabis plant material. In alternative embodiments, the plant or starting material comprises oils or extracts from a trichome or a trichome fraction of a pubescent plant, or an algae or a lichen; and optionally the oils or extracts are from the plant or microbial material by: washing, contacting or exposing the trichome or trichome fraction, or the pubescent plant, algae or lichen, with: at least one non-polar, organic solvent; at least one polar, organic solvent; or, a mix of at least one non-polar, organic solvent with (and) at least one polar, organic solvent.

Chapt. III

In alternative embodiments, provided are methods of and processes for isolating or purifying a cannabinoid or a substantially pure Δ⁸-THC from a natural or a synthetic source, e.g., a plant or a microbial material.

In one embodiment, the method comprises:

-   -   a. providing an extract, aliquot or sample of the natural or         synthetic source comprising at least one of: cannabidiol (CBD),         Δ⁹ THC, Δ⁸ THC, and other cannabinoids from the natural or a         synthetic source, wherein the natural source comprises a plant         or a microbial material;     -   b. providing a continuous chromatography apparatus comprising a         plurality of stations and a plurality of chromatography columns,         wherein optionally the plurality of stations and the plurality         of chromatography columns comprise at least 2, 3, 4, 5, 6, 7, or         8 or more, or between about 3 and 30, stations and/or         chromatography columns;     -   c. introducing or loading the extract, aliquot or sample of the         natural or synthetic source into a first column at a first         station, wherein CBD, Δ⁹ THC, and Δ⁸ THC reversibly binds to the         first column;     -   d. moving the first column to a next or second station;     -   e. eluting the extract from the first chromatography column with         a first elution solvent at the next or second station, wherein         the first elution solvent does not elute the CBD, Δ⁹ THC, and Δ⁸         THC from the first column;     -   f. collecting the first elution fractions from the first column;     -   g. removing all or substantially most of the first elution         solution from the first elution fractions to produce a first         purified extract substantially free of CBD, Δ⁹ THC, and Δ⁸ THC;     -   h. optionally repeating steps d to g with at least one         additional first elution solution;     -   i. when the first column is moved to the next or second station,         introducing or loading the extract into a next or second column         at the first station;     -   j. moving the second column to the next or second station after         the first column has been moved to a next or third station;     -   k. eluting the extract, or material reversibly bound to the         first column with a second elution solution at the next or         second station to generate one or more second elution fractions,         wherein optionally the second elution solution comprises an         organic solvent;     -   l. collecting the second elution fractions from the second         chromatography column at the second station;     -   m. removing all or substantially most of the second elution         solution from the second elution fractions to produce a second         purified extract comprising at least about 75% of CBD, Δ⁹ THC,         and Δ⁸ THC, or between about 60% and 90% of CBD, Δ⁹ THC, and Δ⁸         THC, or between about 50% and 95% of CBD, Δ⁹ THC, and Δ⁸ THC,     -   wherein optionally the elution is by a gradient elution process         and the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids are         separately collected in different or separate gradient elution         fractions,     -   wherein optionally, if the elution is by a gradient elution         process: in a first gradient solvent non-polar compounds and         terpenes are eluted off the column; in a second gradient solvent         cannabidiol (CBD) is eluted off the column; in a third gradient         a CBD/tetrahydrocannabinol (THC) mix is first eluted and then         remaining THC is eluted off the column; in a fourth gradient         remaining cannabinoids are eluted off the column, wherein         optionally the remaining cannabinoids comprise CBC, CBG, CBN;         and, in a fifth gradient solvent all remaining polar compounds         are eluted off the column, wherein the remaining polar compounds         comprises cannabidiolic acid (CBDA) and tetrahydrocannabinolic         acid (THCA);     -   n. optionally repeating steps j to m with at least one second         additional elution solution;     -   o. optionally repeating steps i to m with at least one         additional column;     -   p. recovering the second purified extract, or recovering the         gradient elution fraction, comprising a substantially pure CBD         and the organic solvent, wherein optionally if a gradient         elution is used the CBD is in one or more elution fractions         separate from one or more of the other cannabinoids or polar         compounds;     -   q. optionally removing all or substantially most of the organic         solvent if an organic solvent is used in the gradient elution         fraction;     -   r. optionally adding a second organic solvent to the second         purified extract;     -   s. adding at least one catalyst to the organic phase under an         inert atmosphere to catalyze the conversion of CBD to Δ⁹-THC and         a second order reaction of Δ⁹-THC to Δ⁸-THC;     -   t. optionally controlling the reaction temperature to be in a         range of between about −10° C. to about 40° C. and measuring the         rate of reaction by observing the conversion of CBD to Δ⁹-THC         and second order reaction of Δ⁹-THC to Δ⁸-THC;     -   u. adding a base to a first organic phase when the Δ⁹-THC has         been substantially converted to Δ⁸-THC;     -   v. allowing the mixture to separate into an aqueous phase and a         second organic phase; and     -   w. recovering the second organic phase comprising the converted         Δ⁸-THC.

In some embodiments, the method further comprises repeating steps s to w.

In some embodiments, the method further comprises: repeating steps c to m and wherein the extract, aliquot or sample of the natural or synthetic source in step c is replaced with the Δ⁸-THC as recovered in step w.

In some embodiments, the extract, aliquot or sample of the natural or synthetic source comprises at least 25% CBD by weight, or between about 10% to 40% CBD by weight, or between about 5% to 50% CBD by weight.

In some embodiments, at least one of the first elution solvent, the additional elution solvent, the second elution solvent, and the second additional elution solvent comprises methanol, ethanol, propanol, butanol, chloroform, dichloromethane, dichloroethane, acetic acid, formic acid, oxalic acid, glycolic acid, ammonium hydroxide, water, or combinations thereof.

In some embodiments, the catalyst comprises a Lewis acid or a non-oxidizing acid. In some embodiments, the catalyst comprises p-toluenesulfonic acid

In some embodiments, the base comprises sodium bicarbonate.

In some embodiments, the method further comprises: dissolving, dissolving and/or diluting the extract, aliquot or sample of the natural or synthetic source in a first solvent before introducing or loading the extract, aliquot or sample of the natural or synthetic source into the first chromatography column, or loading the extract, aliquot or sample into the second or additional chromatography columns.

In some embodiments, the first solvent comprises methanol, ethanol, propanol, isobutanol, butanol, isopropanol, tetrahydrofuran, chloroform, methylene dichloride, dichloroethane, water, or combinations thereof.

In some embodiments, the method further comprises: filtering the extract, aliquot or sample of the natural or synthetic source to remove all or substantially most of the material's solids and color bodies before introducing or loading the extract, aliquot or sample of the natural or synthetic source into the first column, or before the initial loading of the extract, aliquot or sample in the second or additional columns.

In some embodiments, at least one of the first chromatography column, the second chromatography column, or the at least one additional chromatography column comprises a normal phase chromatography column, a reverse phase chromatography column, or an ion exchange chromatography column.

In some embodiments, at least one of the organic solvents and the second organic solvent comprises methanol, ethanol, propanol, isobutanol, butanol, isopropanol, tetrahydrofuran, chloroform, methylene dichloride, dichloroethane, water, or combinations thereof.

In some embodiments, the first and second elution solutions are different.

In some embodiments, there are at least 4 columns, at least four elution solutions, and at least four purified extracts.

In some embodiments, the at least four elution solvents are different.

In alternative embodiments, the natural or synthetic material, or the extract, aliquot or sample, is filtered before being loaded on to the column, and optionally the natural or synthetic material, or the extract, aliquot or sample, is mixed with silica, diatomaceous earth (DE), bentonite clay (BC), or equivalents, or mixtures thereof, before filtering, and optionally the natural or synthetic material, or the extract, aliquot or sample, is chilled before being mixed with the silica, DE, BC or equivalents or is chilled before being filtered.

In alternative embodiments, the microbial material comprises or is derived from a bacterium, an algal cell, a lichen or a yeast, or a recombinant bacterium, algal cell, lichen or yeast cell.

In alternative embodiments, the plant material comprises or is derived from a botanical drug substance, including a hemp or a Cannabis plant, wherein optionally the Cannabis plant is a Cannabis sativa, a Cannabis chemovar, or variants thereof, or a decarboxylated Cannabis plant material. In alternative embodiments, the plant or starting material comprises oils or extracts from a trichome or a trichome fraction of a pubescent plant, or an algae or a lichen; and optionally the oils or extracts are from the plant or microbial material by: washing, contacting or exposing the trichome or trichome fraction, or the pubescent plant, algae or lichen, with: at least one non-polar, organic solvent; at least one polar, organic solvent; or, a mix of at least one non-polar, organic solvent with (and) at least one polar, organic solvent.

Chapt. IV

In alternative embodiments, provided are methods of or processes for purifying cannabinoids from a natural or a synthetic source, e.g., a plant or a microbial material.

In one embodiment, the method comprises:

-   -   a. providing an extract, aliquot or sample comprising at least         one of cannabidiol (CBD), Δ⁹ THC, Δ⁸ THC, and other cannabinoids         from the natural or a synthetic source, wherein optionally the         natural or a synthetic source comprises or is derived from a         plant material;     -   b. providing a continuous chromatography apparatus comprising a         plurality of stations and a plurality of chromatography columns,         wherein optionally the plurality of stations and the plurality         of chromatography columns comprise at least 2, 3, 4, 5, 6, 7, or         8 or more, or between about 3 and 30, stations and/or         chromatography columns;     -   c. introducing or loading the extract, aliquot or sample into a         first column at a first station;     -   d. moving the first column to a next or second station and a         second column to the first station;     -   e. eluting the extract, aliquot or sample from the first         chromatography column with a first elution solvent at the next         or second station,     -   wherein optionally the elution is by a gradient elution process         and the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids are         separately collected in different or separate gradient elution         fractions,     -   wherein optionally, if the elution is by a gradient elution         process: in a first gradient solvent non-polar compounds and         terpenes are eluted off the column; in a second gradient solvent         cannabidiol (CBD) is eluted off the column; in a third gradient         a CBD/tetrahydrocannabinol (THC) mix is first eluted and then         remaining THC is eluted off the column; in a fourth gradient         remaining cannabinoids are eluted off the column, wherein         optionally the remaining cannabinoids comprise CBC, CBG, CBN;         and, in a fifth gradient solvent all remaining polar compounds         are eluted off the column, wherein the remaining polar compounds         comprises cannabidiolic acid (CBDA) and tetrahydrocannabinolic         acid (THCA);     -   f. collecting the first elution fractions from the first column         at the second station;     -   g. removing all or substantially most of the first elution         solution from the first elution fractions to produce a first         purified extract, wherein optionally the first purified extract         comprises THC, CBC, CBG, CBN, CBDA and/or THCA, and the THC,         CBC, CBG, CBN, CBDA and/or THCA are eluted separately in         different elution fractions to yield separate purified first         extracts;     -   h. optionally repeating steps d to g with at least one         additional first or other elution solution;     -   i. when the first column is moved to the next or third station,         the second column is moved to the second station where extract         or reversibly bound material is eluted with the first elution         solution, and another batch of the extract, aliquot or sample is         introduced or loaded into a next or third column at the first         station;     -   j. moving the second column to the next or second station;     -   k. eluting the extract from the second column with a first         elution solution at the next or second station;     -   l. collecting the second elution fractions from the second         column at the second station,     -   wherein optionally the elution is by a gradient elution process         and the Δ⁹ THC and/or Δ⁸ THC and/or other cannabinoids are         separately collected in different or separate gradient elution         fractions,     -   wherein optionally, if the elution is by a gradient elution         process: in a first gradient solvent non-polar compounds and         terpenes are eluted off the column; in a second gradient solvent         cannabidiol (CBD) is eluted off the column; in a third gradient         a CBD/tetrahydrocannabinol (THC) mix is first eluted and then         remaining THC is eluted off the column; in a fourth gradient         remaining cannabinoids are eluted off the column, wherein         optionally the remaining cannabinoids comprise CBC, CBG, CBN;         and, in a fifth gradient solvent all remaining polar compounds         are eluted off the column, wherein the remaining polar compounds         comprises cannabidiolic acid (CBDA) and tetrahydrocannabinolic         acid (THCA);     -   m. removing all or substantially most of the second elution         solution from the second elution fractions to produce a second         purified extract;     -   n. optionally repeating steps j to m with at least one second         additional elution solution; and     -   o. optionally repeating steps i to m with at least one         additional column.

In some embodiments, the extract comprises at least 25% CBD by weight, or between about 10% to 40% CBD by weight, or between about 5% to 50% CBD by weight.

In some embodiments, at least one of the first elution solution, the additional elution solution, the second elution solution, and the second additional elution solution comprises methanol, ethanol, propanol, butanol, chloroform, dichloromethane, dichloroethane, acetic acid, formic acid, oxalic acid, glycolic acid, ammonium hydroxide, water, or combinations thereof.

In some embodiments, the method further comprises: dissolving the extract in a first solvent before introducing the extract into the first chromatography column or the second chromatography column.

In some embodiments, the first solvent comprises methanol, ethanol, propanol, isobutanol, butanol, isopropanol, tetrahydrofuran, chloroform, methylene dichloride, dichloroethane, water, or combinations thereof.

In some embodiments, at least one of the first chromatography column, the second chromatography column, or the at least one additional chromatography column comprises a normal phase chromatography column, a reverse phase chromatography column, or an ion exchange chromatography column.

In some embodiments, the first and second elution solutions are different.

In some embodiments, there are at least 2, 3, 4, 5, 6, 7, or 8 or more columns, with at least 2, 3, 4, 5, 6, 7, or 8 or more corresponding elution solvents, and at least 2, 3, 4, 5, 6, 7, or 8 or more corresponding purified extracts.

In some embodiments, the at least 2, 3, 4, 5, 6, 7, or 8 or more elution solutions are different.

In some embodiments, the method further comprises measuring the amount of the cannabinoid in the first or second purified extract.

In alternative embodiments, the natural or synthetic material, or the extract, aliquot or sample, is filtered before being loaded on to the column, and optionally the natural or synthetic material, or the extract, aliquot or sample, is mixed with silica, diatomaceous earth (DE), bentonite clay (BC), or equivalents, or mixtures thereof, before filtering, and optionally the natural or synthetic material, or the extract, aliquot or sample, is chilled before being mixed with the silica, DE, BC or equivalents or is chilled before being filtered.

In alternative embodiments, the microbial material comprises or is derived from a bacterium, an algal cell, a lichen or a yeast, or a recombinant bacterium, algal cell, lichen or yeast cell.

In alternative embodiments, the plant material comprises or is derived from a botanical drug substance, including a hemp or a Cannabis plant, wherein optionally the Cannabis plant is a Cannabis sativa, a Cannabis chemovar, or variants thereof, or a decarboxylated Cannabis plant material. In alternative embodiments, the plant or starting material comprises oils or extracts from a trichome or a trichome fraction of a pubescent plant, or an algae or a lichen; and optionally the oils or extracts are from the plant or microbial material by: washing, contacting or exposing the trichome or trichome fraction, or the pubescent plant, algae or lichen, with: at least one non-polar, organic solvent; at least one polar, organic solvent; or, a mix of at least one non-polar, organic solvent with (and) at least one polar, organic solvent.

Chapt. V

In alternative embodiments, provided are methods of and processes for purifying a cannabinoid from a natural or a synthetic source, e.g., a plant or a microbial material, to a purified mixture comprising Δ⁸tetrahydrocannabinol (Δ⁸-THC) and Δ⁹tetrahydrocannabinol (Δ⁹-THC).

In alternative embodiments, the method comprises:

-   -   a. providing an extract, aliquot or sample comprising at least         one of cannabidiol (CBD), Δ⁹ THC, Δ⁸ THC, and other cannabinoids         from the natural or a synthetic source, wherein optionally the         natural or a synthetic source comprises or is derived from a         plant or a microbial material;     -   b. providing a continuous chromatography apparatus comprising a         plurality of stations and a plurality of chromatography columns,         wherein optionally the plurality of stations and the plurality         of chromatography columns comprise at least 2, 3, 4, 5, 6, 7, or         8 or more, or between about 3 and 30, stations and/or         chromatography columns;     -   c. introducing or loading the extract, aliquot or sample into a         first column at a first station, wherein the at least one of         cannabidiol (CBD), Δ⁹ THC, Δ⁸ THC, and other cannabinoids         reversibly bind to the first column,     -   wherein optionally some of or substantially most of the extract,         aliquot or sample material not reversibly bound to the first         column at the first station is removed with a first wash         solution before the column is moved to a next or a second         station;     -   d. moving the first column to a next or a second station;     -   e. eluting the extract, aliquot or sample from the first         chromatography column with a first elution solution at the next         or second station, wherein the first elution solution does not         elute the CBD, Δ⁹ THC and Δ⁸ THC, which remains on the first         chromatography column;     -   f. collecting the first elution fractions from the first column;     -   g. removing all or substantially most of the first elution         solution from the first elution fractions off the first column         to produce a first purified extract substantially free of CBD,         Δ⁹ THC, and Δ⁸ THC;     -   h. optionally repeating steps d to g with at least one         additional elution solution off the first column;     -   i. when the first column is moved to the next or second station,         a second column is introduced or moved into the first station         where another batch of the extract, aliquot or sample is         introduced or loaded;     -   j. moving the second column to the next or second station when         the first column is moved to a third station;     -   k. eluting the extract or material reversibly bound on the first         column (which comprises at least one of cannabidiol (CBD), Δ⁹         THC, Δ⁸ THC, and other cannabinoids) from the first column with         a second elution solution at the next or second station,     -   wherein optionally the elution using the second elution solution         is by a gradient elution process and the Δ⁹ THC and/or Δ⁸ THC         and/or other cannabinoids are separately collected in different         or separate gradient elution fractions,     -   wherein optionally, if the elution is by a gradient elution         process: in a first gradient solvent non-polar compounds and         terpenes are eluted off the column; in a second gradient solvent         cannabidiol (CBD) is eluted off the column; in a third gradient         solvent a CBD/tetrahydrocannabinol (THC) mix is first eluted and         then remaining THC is eluted off the column; in a fourth         gradient remaining cannabinoids are eluted off the column,         wherein optionally the remaining cannabinoids comprise CBC, CBG,         CBN; and, in a fifth gradient solvent all remaining polar         compounds are eluted off the column, wherein the remaining polar         compounds comprises cannabidiolic acid (CBDA) and         tetrahydrocannabinolic acid (THCA);     -   l. collecting second elution fractions from the first         chromatography column;     -   m. removing all or substantially most of the second elution         solution from the first elution fractions to produce a second         purified extract comprising at least 75% of the total amount of         CBD, Δ⁹ THC, and Δ⁸ THC, or between about 60% and 90% of CBD, Δ⁹         THC, and Δ⁸ THC, or between about 50% and 95% of CBD, Δ⁹ THC,         and Δ⁸ THC;     -   n. optionally repeating steps j to m with at least one second         additional elution solution;     -   o. optionally repeating steps i to m with at least one         additional column;     -   p. mixing the second purified extract with a reaction mixture         comprising an organic solvent and a catalyst;     -   q. reacting the mixture at a controlled temperature for a period         of time, wherein optionally the period of time is between about         30 seconds and 30 minutes;     -   r. adding a base to the mixture;     -   s. separating the mixture into an aqueous phase and an organic         phase, if an organic solvent is used:     -   t. loading the organic phase onto a normal phase chromatography         column;     -   u. eluting the organic phase with a second organic solvent; and     -   v. recovering a third purified extract comprising at least about         30%, 40%, 50% or 60% or more of Δ⁸-THC and Δ⁹-THC.

In some embodiments, the method further comprises repeating steps p to v.

In some embodiments, the base comprises an alkali metal hydrogen carbonate, an alkali metal carbonate, lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)), calcium hydroxide (Ca(OH)), strontium hydroxide (Sr(OH)), barium hydroxide (Ba(OH)).

In some embodiments, the controlled temperature is in a range of −10° C. to 40° C.

In some embodiments, the extract comprises at least 25% CBD by weight, or between about 10% to 40% CBD by weight, or between about 5% to 50% CBD by weight.

In some embodiments, at least one of the first elution solution, the additional elution solution, the second elution solution, and the second additional elution solution comprises methanol, ethanol, propanol, butanol, chloroform, dichloromethane, dichloroethane, acetic acid, formic acid, oxalic acid, glycolic acid, ammonium hydroxide, water, or combinations thereof.

In some embodiments, the organic solvent comprises ethylene dichloride, chloroform, methylene dichloride, toluene, xylene, benzene, hexane, pentane, or combinations thereof.

In some embodiments, the catalyst comprises a Lewis acid or a non-oxidizing acid. In some embodiments, the catalyst comprises p-toluenesulfonic acid

In some embodiments, the method further comprises dissolving, diluting or filtering the extract, aliquot or sample of the natural or synthetic source in a first solvent before introducing or loading the extract, aliquot or sample of the natural or synthetic source into the first chromatography column, or the second or additional chromatography columns at the first station.

In some embodiments, the first solvent comprises methanol, ethanol, propanol, isobutanol, butanol, isopropanol, tetrahydrofuran, chloroform, methylene dichloride, dichloroethane, water, or combinations thereof.

In some embodiments, the method further comprises filtering the extract, aliquot or sample of the natural or synthetic source to remove all or substantially most of the material's solids and color bodies before introducing or loading the extract, aliquot or sample into the first column, or before the initial loading of the extract, aliquot or sample in the second or additional columns at the first station.

In some embodiments, at least one of the first chromatography column, the second chromatography column, or the at least one additional chromatography column comprises a normal phase chromatography column, a reverse phase chromatography column, or an ion exchange chromatography column.

In some embodiments, the method further comprises measuring the amount of Δ⁹-THC in the organic phase.

In some embodiments, the first and second elution solutions are different.

In some embodiments, there are at least 4 columns, at least four elution solutions, and at least four purified extracts.

In some embodiments, the at least four elution solutions are different.

In alternative embodiments, the natural or synthetic material, or the extract, aliquot or sample, is filtered before being loaded on to the column, and optionally the natural or synthetic material, or the extract, aliquot or sample, is mixed with silica, diatomaceous earth (DE), bentonite clay (BC), or equivalents, or mixtures thereof, before filtering, and optionally the natural or synthetic material, or the extract, aliquot or sample, is chilled before being mixed with the silica, DE, BC or equivalents or is chilled before being filtered.

In alternative embodiments, the microbial material comprises or is derived from a bacterium, an algal cell, a lichen or a yeast, or a recombinant bacterium, algal cell, lichen or yeast cell.

In alternative embodiments, the plant material comprises or is derived from a botanical drug substance, including a hemp or a Cannabis plant, wherein optionally the Cannabis plant is a Cannabis sativa, a Cannabis chemovar, or variants thereof, or a decarboxylated Cannabis plant material. In alternative embodiments, the plant or starting material comprises oils or extracts from a trichome or a trichome fraction of a pubescent plant, or an algae or a lichen; and optionally the oils or extracts are from the plant or microbial material by: washing, contacting or exposing the trichome or trichome fraction, or the pubescent plant, algae or lichen, with: at least one non-polar, organic solvent; at least one polar, organic solvent; or, a mix of at least one non-polar, organic solvent with (and) at least one polar, organic solvent.

The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of embodiments as provided herein will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 schematically illustrates an exemplary method as provided herein using a continuous chromatography apparatus comprising a plurality of stations and a plurality of chromatography columns.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Chapter I

In alternative embodiments, provided are continuous isolation and purification processes for preparing a substantially pure cannabidiol or a product enriched in cannabidiol from natural or synthetic sources, including plant or microbial material extracts. In alternative embodiments, provided herein are improved methods for converting cannabidiol (CBD) to Δ⁸-THC and Δ⁹-THC, including a purification and conversion process based on a simple combination of continuous chromato-graphic gradient elutions and semi continuous isomerization reactions. This exemplary process is simple, efficient and economic.

In alternative embodiments, provided are methods of preparing cannabinoids in substantially pure form starting from plant extract material and conversion of the purified CBD to form both Δ⁸-THC and Δ⁹-THC and subsequent purification of the produced Δ⁸-tetrahydrocannabinol into Δ⁹-tetrahydrocannabinol using continuous chromatography.

In alternative embodiments, provided are processes for producing and isolating cannabinoids from Cannabis and hemp extracts which contain cannabinoids in minute amounts. In alternative embodiments, provided are processes for producing and isolating cannabinoids from natural materials, including plant or plant extracts, microbes, or botanical drug substances, or synthetically and semi-synthetically prepared cannabinoid products, or from recombinantly engineered microbes, e.g., yeasts or bacteria recombinantly engineered to express one or more cannabinoids. In one embodiment, exemplary methods are inexpensive and provide specific cannabinoid concentrates (e.g., of CBD, Δ⁸-THC, Δ⁹-THC) of high purity.

In one embodiment, exemplary methods provide a simple and economical continuous process for separating and concentrating cannabinoids from solvent-extracted cannabinoid containing materials. In one embodiment, exemplary methods provide a method that first converts the substantially isolated CBD into a mixture of Δ⁸-THC and Δ⁹-THC, and then subsequently purifies and/or isolates the Δ⁸-THC and Δ⁹-THC. In alternative embodiments, the solvent-extracted cannabinoid containing materials are derived from synthetic or biological materials such as hemp and Cannabis or botanical drug substances, or from microbial materials; and the solvent extraction methods can be polar solvent extractions, nonpolar solvent extractions, or the solvent extraction methods can comprise use of super critical carbon dioxide or mixtures thereof. The solvent extraction methods can extract cannabinoids substantially from the synthetic or biological, e.g., plant, matter, along with other plant matter comprising lipids, waxes, monoterpenes, sesquiterpenes, hydrocarbons, alkaloids, flavonoids and chlorophylls.

In alternative embodiments, methods provided herein comprise subjecting cannabinoid containing solvent extract starting materials to a number of chromatographic resins in various contacting steps using various gradient elution solutions.

In alternative embodiments, the cannabinoids which can be fractionated and isolated using methods as provided herein, or which can be produced in reactions as provided herein, or from which the solvent extracts are derived can be from, or can comprise: Δ⁸tetrahydrocannabinol (Δ⁸ THC); Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetrahydrocannabidiol (THCBD); tetrahydrocannabigerol (THCBG); tetrahydrocannabichromene (THCBC); tetrahydrocannabidivarol (THCBDV), or combinations thereof, including carboxylic acid precursors of the foregoing compounds and related naturally occurring compounds and their derivatives.

In alternative embodiments, provides are methods of preparing or obtaining a substantially pure cannabinoid or a product enriched in a given cannabinoid comprising:

-   -   (i) obtaining an extract or extract solution containing a         cannabinoid or a cannabinoid acid from a synthetic or         biological, e.g., plant, material;     -   (ii) optionally filtering the extract of step (i) to remove all         or substantially all solids and color bodies;     -   (iii) removing the extract solvent (as an extract fraction);     -   (iv) continuously loading of an amount of extract solution over         a defined time increment over multiple stationary phase resins         columns such as a normal phase, reverse phase and/or ion         exchange chromatographic resin,     -   e.g., at first station;     -   (v) continuously eluting the extract solution using multiple         defined gradient elution solutions at specific time increments         and volumes, e.g., at a second station;     -   (vi) continuously collecting the gradient elution fractions;     -   (vii) removal of the gradient elution solution from the produced         fractions to generate a substantially purified extract;     -   (viii) optionally loading of specific first gradient elution         fractions on a reverse phase, ion exchange or normal         chromatographic resin;     -   (ix) continuously eluting the first gradient elution fractions         with a second gradient elution solvent; and,     -   (x) removing of the second gradient elution solvent from the         produced fractions to produce a purified extract.

In alternative embodiments, the methods further comprise:

a step (xi): loading of first and second gradient elution solvents onto an ion exchange chromatographic resin;

a step (xii): eluting an extract solution from the ion exchange chromatographic resin of step (x) using a gradient solvent and collecting the gradient elution fractions; and

-   -   (xiii) removing the gradient elution solvent from the produced         fractions of step (xii) to produce purified and substantially         purified extract.

In alternative embodiments, provided are methods of substantially converting CBD to Δ⁸-THC and Δ⁹-THC comprising:

-   -   (a) providing a reaction mixture comprising a catalyst in an         organic solvent;     -   (b) adding CBD material, e.g., a CBD purified or isolated using         steps (vi) and steps (xiii), above;     -   (c) mixing said reaction mixture;     -   (d) reacting mixture for a period of time at a controlled         temperature;     -   (e) adding a base to the reaction mixture;     -   (f) allowing the mixture to separate into an aqueous phase and         an organic phase;     -   (g) removing the organic phase; and,     -   (h) loading the organic phase onto a normal phase chromatography         column     -   (i) eluting the organic phase with an organic solvent and         recovering substantially pure CBD, Δ⁸ THC and Δ⁹ THC     -   optionally, repeating steps (a) through (i).

In alternative embodiments, a “substantially pure” preparation of cannabinoid is defined as a preparation having a chromatographic purity (of the desired cannabinoid or cannabinoid acid) of greater than about 75%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99%, or greater than about 99.5%, or between about 70% and 99.9%, as determined by area normalization of an HPLC profile.

In alternative embodiments, the term “product enriched in a given cannabinoid” encompasses preparations having at least about 50%, or greater than about 75%, or greater than about 90%, 95% or 98%, or between about 50% and 99.9%, chromatographic purity, for the desired cannabinoid.

In alternative embodiments, the term “about” is within 20% of the stated value, or 19%, or 18%, or 17%, or 16%, or 15%, or 14%, or 13%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, or 0.5%, or 0.1%, or 0.05%, or 0.01%, or is between 20% and 0.01% of the stated value.

In alternative embodiments, a non-purified, or non-substantially purified, product can comprise a greater proportion of impurities, non-target materials and/or other cannabinoids than a “substantially pure” preparation. The cannabinoid can be (e.g., a cannabinoid purified or isolated by, or made by a reaction of, a method as provided herein can be): Δ⁸-tetrahydrocannabinol (Δ⁸-THC); Δ⁹-tetrahydrocannabinol (Δ⁹-THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetra-hydrocannabidiol (THCBD); tetra-hydrocannabigerol (THCBG); tetra-hydrocannabichromene (THCBC); or, tetra-hydrocannabidivarol (THCBDV); a carboxylic acid precursor of the foregoing compounds; and related naturally occurring compounds and their derivatives.

In alternative embodiments, the term “cannabinoids”, e.g., a cannabinoid purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,1′-di-methyl-pyrane ring, a variedly derivatized aromatic ring and/or a variedly unsaturated cyclohexyl ring and their immediate chemical precursors.

In alternative embodiments, the term “cannflavins”, e.g., a cannflavin purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,4-pyrone ring fused to a variedly derivatized aromatic ring and linked to a second variedly derivatized aromatic ring.

In alternative embodiments, the term “Lewis acid” refers to a powerful electron pair acceptor; and examples include but are by no means limited to BF₃Et₂O (boron trifluoride diethyl etherate), p-toluenesulfonic acid and boron trifluoride.

In alternative embodiments, the term “non-oxidizing acid” refers to hydrobromic, hydrochloric, hydrofluoric, acetic, benzoic, chloroacetic, formic, phosphoric, sulfuric, trifluroacetic and oxalic acids.

In alternative embodiments, the term “essential oils”, e.g., an essential oil used as a starting material in a process as provided herein, or an essential oil that may be isolated by a process as provided herein, includes or refers to a family of natural products that can contain a multiple of the 5-membered isoprene unit variedly substituted, often cyclized to form one or more ring systems; they can also contain series of aldehydes and/or ketones and esters of a variety of carboxylic acid substituted compounds.

In alternative embodiments, provided are methods for extracting and/or purifying cannabinoids from any natural or synthetic sources, including plant or microbial material or extracts known to contain such cannabinoids, cannflavins and essential oils; and, optionally to purify cannflavins and to optionally purify essential oils. In alternative embodiments, the extract is passed through a series of chromatographic columns, for example, a normal phase column, a reversed phase column or an ion exchange column as a continuous simulated moving bed configuration.

In one embodiment, the chromatographic column is arranged for gradient elution fractioning using normal phase, reverse phase and/or ion exchange chromatography. In one embodiment Δ⁹-THC and CBD are fractionated out of the eluent. For example, in one embodiment, as the extract is passed over the column, Δ⁹-THC and CBD are differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract comes off the column after gradient elution transition, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THC and CBD. The fractions free of Δ⁹-THC and CBD are pooled, thereby producing an extract with Δ⁹-THC and CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, (substantially) only Δ⁹-THC is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially retained or detained (e.g., reversibly bound) on the column. As the extracts pooled from previous elution fractions come off the column, the initial fractions eluted off the column will be free of Δ⁹-THC. These fractions free of Δ⁹-THC are pooled, thereby producing an extract with Δ⁹-THC substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged, for example, 3, 4, 5, 6, 7, 8, 9, or 10 or more, or between 3 and 30, columns are arranged in a continuous rotation traveling through a series of contact points where gradient elution solutions and extract solution are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent is introduced, the second column moves to the fifth position where the fourth gradient solvent is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced. In a particular embodiment, the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent.

For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment, second gradient solvent elutes CBD, which is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment, the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent.

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged, for example, 3, 4, 5, 6, 7, 8, 9, or 10 or more, or between 3 and 30, columns are arranged in a continuous rotation traveling through a series of contact points where gradient elution solvents and extract solution are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution solvent is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced, and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced, and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent is introduced, the second column moves to the fifth position where the fourth gradient is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced. In a particular embodiment the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment, the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent. The second gradient solvent n containing CBD is combined with a Lewis acid or non-oxidizing acid catalyst, optionally additional CBD or substantially pure CBD and/or Δ8-tetrahydrocannabinol (Δ8-THC) and/or Δ9-tetrahydrocannabinol (Δ9-THC) can be added to the reaction mixture, mixing said reaction mixture for a period of time; adding a neutralizing agent to said mixture; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally adding organic phase to a chromatography column and eluting the (Δ8-THC) and/or Δ9-tetrahydrocannabinol (Δ9-THC) from the organic phase. The tetrahydrocannabinol can then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase.

In some embodiments, (substantially) only CBD is substantially fractionated out of the eluent. That is, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of CBD. These fractions free of CBD are pooled, thereby producing an extract with CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, Δ⁹-THC carboxylic acid species or Δ⁹-tetra-hydrocannabinolic acid (THCA) and the CBD carboxylic acid species cannabidiolic acid (CBDA) are fractionated out of out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a first elution are passed over the column, Δ⁹-THCA and CBDA are differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled first elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THCA and CBDA. These fractions (substantially) free of Δ⁹-THCA and CBDA are pooled, thereby producing an extract with Δ⁹-THCA and CBDA substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, the Δ⁹-THC can be eluted from the column, extracted or concentrated, for purifying, or substantially purifying, Δ⁹-THC. In alternative embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) a specific cannabinoid or groups of cannabinoids or their carboxylic acid species, cannflavin or essential oil or class of cannabinoids, cannflavins or essential oils out of the eluent, for example, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabidivarol (CBDV), tetrahydrocannabidiol (THCBD), tetrahydrocannabigerol (THCBG), tetrahydrocannabichromene (THCBC), tetrahydrocannabidivarol (THCBDV), Δ⁸-THC, the carboxylic acid precursors of the foregoing compounds, and related naturally occurring compounds and their derivatives. In alternate embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) cannflavins and related naturally occurring compounds and their derivatives. In alternate embodiments, the system is arranged to fractionate the components of essential oils. The list of compounds provided herein is not exhaustive and is in no way intended to be limiting. In these embodiments, the compound(s) of interest are retained or detained (e.g., reversibly bound) on the column so that fractions (alternatively, the last fractions) of the extract eluted from the column contain the compounds(s) of interest. In alternative embodiments, fractions containing the compound(s) of interest are pooled. In some embodiments, different compounds can be extracted with different solvents and then combined into a single extract. As will be appreciated by one knowledgeable in the art, in this manner, several different cannabinoids could be purified from a single extract.

In alternative embodiments, the “plant material” or “plant extracts” comprise or are derived from one or more Cannabis or hemp plants, or from other plants. In alternative embodiments, the microbial material or extracts comprise or are derived from yeast, lichen, algae or bacteria. The term “plant material” encompasses a plant or plant part (e.g. bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries or parts thereof) as well as exudates, and includes material falling within the definition of “botanical raw material” in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research. The term “Cannabis plant(s)” encompasses wild type Cannabis sativa and also variants thereof, including Cannabis chemovars (varieties characterized by virtue of chemical composition) which naturally contain different amounts of the individual cannabinoids, also Cannabis sativa subspecies indica including the variants var. indica and var. kafiristanica, Cannabis indica and also plants which are the result of genetic crosses, self-crosses or hybrids thereof. The term “Cannabis plant material” also can encompass plant material derived from one or more Cannabis plants, and can comprise any “Cannabis plant material” including, e.g., herbal Cannabis and dried Cannabis biomass. The term “Cannabis plant material” also can encompass “decarboxylated Cannabis plant material”, which refers to Cannabis plant material which has been subject to a decarboxylation step in order to convert cannabinoid acids to the corresponding free cannabinoids. In alternative embodiments, a starting material for a purification process as provided herein is an extract solution containing a cannabinoid or cannabinoid acid obtained from a natural or a synthetic source, e.g., a plant or microbial material. In alternative embodiments, the “extract solution containing a cannabinoid or cannabinoid acid” comprises a solvent extract of a plant or microbial material. Solvents used for extraction for use in the preparation of extract solutions can comprise non-polar solvents, polar solvents such as ethanol, methanol or water, or organic solvents such as liquid carbon dioxide, and combinations thereof. The solvent can be an organic solvent, selected from the group consisting of: non-polar solvents include liquid non-polar solvents comprising lower C1-C12, preferably C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol, methanol, water; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; or a supercritical fluid such as CO₂ with or without an organic solvent modifier.

In alternative embodiments, the extract is prepared by dissolving or partially dissolving the natural or synthetic, or the plant or microbial material in a solvent, removing insoluble material from the resultant solution (optionally by filtration with or without activated carbon, precipitation, centrifugation and the like), and optionally removing some or all of the extraction solvent from the solution (optionally by rotary evaporation) to form an extract or extract solution or concentrate containing a cannabinoid or cannabinoid acid.

In alternative embodiments, the plant or microbial material or extracts used as a starting material comprise oils or extracts from a trichome or a trichome fraction of a pubescent plant, or an algae or a lichen. The oils or extracts can be “harvested” from the plant or microbial material by: washing, contacting or exposing the trichome or trichome fraction, or the pubescent plant, algae or lichen, with: at least one non-polar, organic solvent; at least one polar, organic solvent; or, a mix of at least one non-polar, organic solvent with (and) at least one polar, organic solvent, and collecting or separating the solvent from the trichome or trichome fraction, or the pubescent plant, algae or lichen, wherein the solvent comprises the oil or extract to be further processed using a method as provided herein. In alternative embodiments, the pubescent plant or plant part, the algae or algae part, or the lichen or lichen part, other than harvesting or collecting, is unprocessed or has not been physically processed, or has not been exposed to: a solvent or aqueous solution; a heating or drying process; a dehydration process; a crushing process; and/or, a chopping, macerating or mincing process, since (after) its harvest, before the trichome or trichome fraction, or the pubescent plant, algae or lichen is washed with, contacted by or exposed to: at least one non-polar, organic solvent; at least one polar, organic solvent; or, a mix of at least one non-polar, organic solvent with (and) at least one polar, organic solvent.

In alternative embodiments, extractions comprise using a technique referred to as accelerated solvent extraction, which can use subcritical water or any combination of water and solvent. In one embodiment, when isolating cannabinoid acids, a modified pH gradient elution solvent is used. The primary purpose of this pH adjustment (the modified pH gradient) is to promote or prevent ionization of the cannabinoid acid. pH modified gradient elution solvents s may be achieved by the additional of a small volume of acid or base to the solvent. It may be sufficient to add a relatively weak acid, such as acetic acid, oxalic acid, glycolic acid, carbonic acid or ammonium hydroxide or a small amount of base or buffering agent such as sodium hydroxide, magnesium hydroxide, sodium carbonate or sodium bicarbonate. For any given purification process the optimal amount and type of acid or base used may be determined empirically. An alternative exemplary acidified solvent is 0.1% acetic acid in ethanol or 0.1% sodium hydroxide in ethanol. In alternative embodiments, the neutralizing agent consists of for example sodium hydroxide, sodium carbonate, potassium carbonate, and potassium t-amylate, sodium bicarbonate.

Acidified non-polar and polar solvents of the types described above can be useful in preparation of gradient elutions using ion exchange chromatography. The solvents used in the conversion can comprise an organic solvent, e.g., a non-polar solvent, including a liquid non-polar solvent comprising lower C1-C12, or C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane, hexane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; a heterocyclic compound or cyclic ether for example, tetrahydrofuran and 2-methyltetrahydrofuran and aromatic ring hydrocarbons such as benzene, toluene, xylene and ethylbenzene.

In alternative embodiments, the plant material is subjected to a decarboxylation step prior to solvent extraction. The purpose of the decarboxylation step is to convert cannabinoid acids present in the plant or microbial material to the corresponding free cannabinoids. In alternative embodiments, the decarboxylation is carried out by heating the plant or microbial material to a defined temperature for a suitable length of time. Decarboxylation of cannabinoid acids is a function of time and temperature, thus at higher temperatures a shorter period of time will be taken for complete decarboxylation of a given amount of cannabinoid acid. In alternative embodiments selecting appropriate conditions for decarboxylation consideration includes minimizing thermal degradation of the desirable, pharmacological cannabinoids into undesirable degradation products, particularly thermal degradation of Δ⁹-THC. In alternative embodiments, the decarboxylation is carried out in a multi-step heating process in which the plant or microbial material is: i) heated to a first temperature for a first (relatively short) time period to evaporate off retained water and allow for uniform heating of the plant or microbial material; and ii) the temperature is increased to a second temperature for a second time period (typically longer than the first time period) until at least 95% conversion of the acid cannabinoids to their neutral form has occurred.

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” prepared from the starting plant or microbial material comprises a “botanical drug substance” prepared from the plant or microbial material, or a polar or non-polar solvent solution of such a botantical drug substance. In the context of this application a “botanical drug substance” is an extract derived from plant or microbial material, which extract fulfills the definition of “botanical drug substance” provided in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research of: “A drug substance derived from one or more plants, algae, or macroscopic fungi. It is prepared from botanical raw materials by one or more of the following processes: pulverization, decoction, expression, aqueous extraction, ethanolic extraction, or other similar processes.” “Botanical drug substances” derived from Cannabis plants include primary extracts prepared by such processes as, for example, maceration, percolation, and solvent extraction.

In alternative embodiments, solvent extraction may be carried out using essentially any solvent that dissolves, or substantially dissolves, cannabinoids/cannabinoid acids, such as for example C1 to C5 alcohols (e.g. ethanol, methanol), C5-C12 alkanes (e.g. hexane), norflurane (HFA134a), 1,1,1,2,3,3,3-Heptafluoropropane (or HFA227), chloroform, dichloromethane, dichloroethane and carbon dioxide. When solvents such as those listed above are used, the resultant extract typically contains non-specific lipid-soluble material. This can optionally be removed by a variety of processes including filtration to remove solids, “winterization”, which involves for example chilling to −20° C. or lower followed by filtration to remove waxy ballast, extraction with liquid carbon dioxide and by distillation.

In alternative embodiments, any protocol for the preparation of botanical drug substances from Cannabis and hemp plant material can be used, e.g., as described in International patent application WO 02/064109. In alternative embodiments, the botanical drug substance is obtained by carbon dioxide (CO₂) extraction, polar solvent extraction or non-polar solvent extraction or combinations thereof followed by a filtration. Optionally a secondary extraction is performed to remove a substantial proportion of non-cannabinoid materials, e.g. waxes, wax esters and glycerides, unsaturated fatty acid residues, terpenes, carotenes, and flavonoids and other ballast.

In alternative embodiments, if it is intended to prepare free cannabinoids from the Cannabis plant or microbial material, then the material is heated to a defined temperature for a defined period of time in order to partially or substantially decarboxylate cannabinoid acids to free cannabinoids prior to extraction of the botanical drug substance. In alternative embodiments, the botanical drug substance is prepared according to a process comprising the following steps: i) optional decarboxylation of the plant or microbial material, ii) extraction with polar or non-polar solvent, to produce a crude botanical drug substance, iii) optional precipitation with C1-C5 alcohol to reduce the proportion of non-target materials, iv) removal of the precipitate (preferably by filtration), v) optional treatment with activated charcoal, and vi) evaporation to remove C1-C5 alcohol and water, thereby producing a final botanical drug substance.

In alternative embodiments, provided are methods for converting substantially purified cannabidiol (CBD) to Δ8-tetrahydrocannabinol (Δ8-THC) and Δ9-tetrahydrocannabinol (Δ9-THC). As will be appreciated by one knowledgeable in the art and as discussed below, the reaction times may be varied somewhat, producing product at different yields and purities. Furthermore, functional equivalents may be substituted where appropriate.

In alternative embodiments, an exemplary method of converting CBD to Δ8-tetrahydrocannabinol (Δ8-THC) and Δ9-tetrahydrocannabinol (Δ9-THC) comprises: providing a reaction mixture comprising a Lewis acid or non-oxidizing acid catalyst in a reaction solvent, adding a substantially pure CBD or substantially pure CBD and/or (Δ8-THC) and/or Δ9-tetrahydrocannabinol (Δ9-THC) to the reaction mixture, mixing said reaction mixture for a period of time; adding a neutralizing agent to said mixture; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally removing the reaction solvent; optionally dissolving organic phase in a second solvent; adding organic phase to a chromatography column and eluting the tetrahydrocannabinol from the organic phase. The tetrahydrocannabinol may then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase.

In alternative embodiments, the tetrahydrocannabinol at therapeutically effective concentrations or dosages is combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, methacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients that can be used include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethyl cellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethycellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example, Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed.

In some embodiments, the catalyst is a Lewis acid, for example, p-toluenesulfonic acid, boron trifluoride or BF₃Et₂O. In some embodiments, the BF₃Et₂O (boron trifluoride diethyl etherate) is in dry methylene chloride, ethyl acetate, ethanol, hexane or other organic solvent. In yet other examples, the catalyst may be hydrochloric acid in ethanol or sulfuric acid in cyclohexane.

In some embodiments, the catalyst is a non-oxidizing acid, for example, formic acid, acetic acid or hydrobromic acid. In some embodiments, the non-oxidizing acid is in dry methylene chloride, ethyl acetate, ethanol, hexane or other organic solvent.

In some embodiments, a base is added to the reaction mixture prior to optionally allowing the reaction mixture to separate into organic and aqueous phases. The base may be an alkali metal hydrogen carbonate, carbonate of an alkali metal, lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)), calcium hydroxide (Ca(OH)), strontium hydroxide (Sr(OH)), barium hydroxide (Ba(OH)).

In some embodiments, the organic layer is dried prior to eluting. In these embodiments, a suitable drying or dehydration agent, for example, MgSO4 or Na₂SO₄ is used.

In yet other embodiments, the process may be carried out under an inert atmosphere such as a nitrogen (e.g., N₂) atmosphere.

In alternative embodiments, and as discussed below, yield is determined by looking at the peak area for the isolated compound in the gas chromatography-mass spectra analysis of the crude reaction product mixture and the final reaction product mixture. It is important to note that in the prior art, yield is often calculated on the basis of first isolated crude product before final purification. In some embodiments of processes provided herein yield of Δ8-THC and Δ9-THC is at least about 75%; in other embodiments, the yield of Δ8-THC and Δ9-THC is at least about 90%; and in other embodiments, yield of Δ8-THC and Δ9-THC is at least about 98%; and in yet other embodiments, yield of Δ8-THC and Δ9-THC is between about 75 to 98% or 99%.

In alternative embodiments, purity is determined by Gas Chromatography-Mass Spectrometry (GC-MS) and/or by analytical high-performance liquid chromatography (HPLC). The total ion chromatogram from the GC-MS gives information similar to that provided by a flame ionization detector (FID)-GC in that the peak area is proportional to the mass of the analytes detected. Total peak area and the peak areas of the individual analytes can be compared in the GC-MS case as long as the masses are in generally the same range. As discussed below, in some embodiments, purity of the Δ8-THC and Δ9-THC mixture isolated by the process is greater than about 90%, 95%, 97% or 98%, or purity is greater that about 98% to 99%.

Chapter II

In alternative embodiments, provided are continuous isolation and purification processes for preparing a substantially pure cannabidiol or a product enriched in cannabidiol from synthetic or natural sources, e.g., from plant or microbial material or microbial extracts. In alternative embodiments, provided herein are improved methods for converting cannabidiol (CBD) to Δ⁹-THC, including a purification and conversion process based on a simple combination of continuous chromatographic gradient elutions and semi continuous isomerization reactions. This exemplary process is simple, efficient and economic.

In alternative embodiments, provided are methods of preparing cannabinoids in substantially pure form starting from plant extract material and conversion of the purified CBD to form both Δ⁸ THC and Δ⁹ THC and subsequent conversion of the formed Δ⁸ tetrahydrocannabinol into Δ⁹ tetrahydrocannabinol.

In alternative embodiments, provided are processes for producing and isolating cannabinoids from Cannabis and hemp extracts which contain cannabinoids in minute amounts. In alternative embodiments, provided are processes for producing and isolating cannabinoids from natural materials, including plant or plant extracts, microbes, or botanical drug substances, or synthetically and semi-synthetically prepared cannabinoid products, or from recombinantly engineered microbes, e.g., yeasts or bacteria recombinantly engineered to express one or more cannabinoids. In one embodiment, exemplary methods are inexpensive and provide specific cannabinoid concentrates (e.g., of CBD, Δ⁸-THC, Δ⁹-THC) of high purity.

In one embodiment, exemplary methods provide a simple and economical continuous process for separating and concentrating cannabinoids from solvent-extracted cannabinoid containing materials. In one embodiment, exemplary methods provide a method that first converts the substantially isolated CBD into a mixture of Δ⁸-THC and Δ⁹-THC, and then subsequently converts the Δ⁸-THC into Δ⁹-THC. In alternative embodiments, the solvent-extracted cannabinoid containing materials are derived from synthetic or biological materials such as hemp and Cannabis or botanical drug substances, or from microbial materials; and the solvent extraction methods can be polar solvent extractions, nonpolar solvent extractions, or the solvent extraction methods can comprise use of super critical carbon dioxide or mixtures thereof. The solvent extraction methods can extract cannabinoids substantially from the synthetic or biological, e.g., plant, matter, along with other plant matter comprising lipids, waxes, monoterpenes, sesquiterpenes, hydrocarbons, alkaloids, flavonoids and chlorophylls.

In alternative embodiments, methods provided herein comprise subjecting cannabinoid containing solvent extract starting materials to a number of chromatographic resins in various contacting steps using various gradient elution solvents s.

In alternative embodiments, the cannabinoids which can be fractionated and isolated using methods as provided herein, or which can be produced in reactions as provided herein, or from which the solvent extracts are derived can be from, or can comprise: Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetrahydrocannabidiol (THCBD); tetrahydrocannabigerol (THCBG); tetrahydrocannabichromene (THCBC); tetrahydrocannabidivarol (THCBDV), or combinations thereof, including carboxylic acid precursors of the foregoing compounds and related naturally occurring compounds and their derivatives.

In alternative embodiments, provides are methods of preparing or obtaining a substantially pure cannabinoid or a product enriched in a given cannabinoid comprising:

-   -   (i) obtaining an extract or extract solution comprising or         containing a cannabinoid or a cannabinoid acid from a natural or         a synthetic source,     -   e.g., a plant or microbial material;     -   (ii) filtering the extract or extract solution of step (i) to         remove solids and color bodies;     -   (iii) removing the extract solvent (as an extract fraction);     -   (v) continuously loading of an amount of extract solution over a         defined time increment over multiple stationary phase resins         columns such as a normal phase, reverse phase and/or ion         exchange chromatographic resin;     -   (vi) continuously eluting the extract solution using multiple         defined gradient elution solvents s at specific time increments         and volumes;     -   (vii) continuously collecting the gradient elution fractions;     -   (viii) removal of the gradient elution solvent from the produced         fractions to generate a substantially purified extract     -   (ix) optionally loading of specific first gradient elution         fractions on a reverse phase, ion exchange or normal         chromatographic resin;     -   (x) continuously eluting the first gradient elution fractions         with a second gradient elution solvent; and,     -   (xi) removing of the second gradient elution solvent from the         produced fractions to produce a purified extract.

In alternative embodiments, the methods further comprise:

-   -   a step (xii): loading of first and second gradient elution         solvents s onto an ion exchange chromatographic resin;     -   a step (xiii): eluting an extract solution from the ion exchange         chromatographic resin of step (xi) using a gradient solvent and         collecting the gradient elution fractions; and     -   (xiv) removing the gradient elution solvent from the produced         fractions of step (xiii) to produce purified and substantially         purified extract.

In alternative embodiments, provided are methods of converting CBD to Δ⁸THC and Δ⁹ THC comprising:

-   -   (a) providing a reaction mixture comprising a catalyst in an         organic solvent;     -   (b) adding CBD material from steps (viii) and steps (xiii),         above;     -   (c) mixing said reaction mixture;     -   (d) allowing the mixture to separate into an aqueous phase and         an organic phase;     -   optionally adding a second solvent; and     -   (e) removing the organic phase; and,     -   optionally, repeating steps (a) through (e).

In alternative embodiments, provided are methods for purifying Δ⁸-THC and Δ⁹-THC comprising:

-   -   (a) providing a reaction mixture comprising a stabilizing agent         in an organic solvent;     -   (b) adding Δ⁸-THC and Δ⁹-THC to the reaction mixture;     -   (c) mixing said reaction mixture to form         9-chlorohexahydrocannabinol;     -   (d) combining said reaction mixture with a second organic         solvent;     -   (e) adding an elimination catalyst or reagent to the reaction         mixture to cause an elimination refluxing said reaction mixture         under an inert atmosphere;     -   (f) pouring the mixture into cold water;     -   (g) mixing the mixture;     -   (h) allowing the mixture to separate into an aqueous phase and         an organic phase;     -   optionally adding a second solvent;     -   (i) removing the organic phase, which comprises a purified, or a         substantially pure, Δ⁸-THC and Δ⁹-THC; and     -   optionally, repeating steps (a) through (i).

In alternative embodiments, provided are methods for converting CBD to substantially pure Δ⁹-THC comprising:

-   -   (a) providing a reaction mixture comprising a CBD in an organic         solvent;     -   (b) adding a catalyst to the reaction mixture under a nitrogen         atmosphere;     -   (c) stirring the reaction mixture;     -   (d) adding NaHCO₃ or equivalent to the reaction mixture;     -   (e) allowing the mixture to separate into an aqueous phase and         an organic phase;     -   optionally adding a second solvent;     -   (f) removing the organic phase, which comprises the converted         Δ⁹-THC; and     -   optionally repeating steps (a) through (f).

In alternative embodiments, a “substantially pure” preparation of cannabinoid is defined as a preparation having a chromatographic purity (of the desired cannabinoid or cannabinoid acid) of greater than about 75%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99%, or greater than about 99.5%, or between about 70% and 99.9%, as determined by area normalisation of an HPLC profile.

In alternative embodiments, the term “product enriched in a given cannabinoid” encompasses preparations having at least about 50%, or greater than about 75%, or greater than about 90%, 95% or 98%, or between about 50% and 99.9%, chromatographic purity, for the desired cannabinoid.

In alternative embodiments, a non-purified, or non-substantially purified, product can comprise a greater proportion of impurities, non-target materials and/or other cannabinoids than a “substantially pure” preparation. The cannabinoid can be (e.g., a cannabinoid purified or isolated by, or made by a reaction of, a method as provided herein can be): Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetra-hydrocannabidiol (THCBD); tetra-hydrocannabigerol (THCBG); tetra-hydrocannabichromene (THCBC); or, tetra-hydrocannabidivarol (THCBDV); the carboxylic acid precursors of the foregoing compounds; and related naturally occurring compounds and their derivatives.

In alternative embodiments, the term “cannabinoids” e.g., a cannabinoid purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,1′-di-methyl-pyrane ring, a variedly derivatized aromatic ring and/or a variedly unsaturated cyclohexyl ring and their immediate chemical precursors.

In alternative embodiments, the term “cannflavins”, e.g., a cannflavin purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,4-pyrone ring fused to a variedly derivatized aromatic ring and linked to a second variedly derivatized aromatic ring.

In alternative embodiments, the term “Lewis acid” refers to a powerful electron pair acceptor; and examples include but are by no means limited to BF₃Et₂O (boron trifluoride ditheyl etherate), p-toluenesulfonic acid and boron trifluoride.

In alternative embodiments, the term “non-oxidizing acid” refers to hydrobromic, hydrochloric, hydrofluoric, acetic, benzoic, chloroacetic, formic, phosphoric, sulfuric, trifluroacetic and oxalic acids.

In alternative embodiments, the term “stabilizing agent” refers to a zinc chloride, hydrogen chloride, methylene chloride, simonkolleite, and mixtures thereof; and examples include but are by no means limited to zinc chloride, hydrogen chloride, and simonkolleite, and polymorphs and hydrates thereof.

In alternative embodiments, the term “essential oils”, e.g., an essential oil used as a starting material in a process as provided herein, or an essential oil that may be isolated by a process as provided herein, includes or refers to a family of natural products that can contain a multiple of the 5-membered isoprene unit variedly substituted, often cyclized to form one or more ring systems; they may also contain series of aldehydes and/or ketones and esters of a variety of carboxylic acid substituted compounds.

In alternative embodiments, provided are methods for extracting and/or purifying cannabinoids from any plant material extract known to contain such cannabinoids, cannflavins and essential oils; and, optionally to purify cannflavins and to optionally purify essential oils. In alternative embodiments, the extract is passed through a series of chromatographic columns, for example, a normal phase column, a reversed phase column or an ion exchange column as a continuous simulated moving bed configuration.

In one embodiment, the chromatographic column is arranged for gradient elution fractioning using normal phase, reverse phase and/or ion exchange chromatography. In one embodiment Δ⁹-THC and CBD are fractionated out of the eluent. For example, in one embodiment, as the extract is passed over the column, Δ⁹-THC and CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THC and CBD. The fractions free of Δ⁹-THC and CBD are pooled, thereby producing an extract with Δ⁹-THC and CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, (substantially) only Δ⁹-THC is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extracts are pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be free of Δ⁹-THC. These fractions free of Δ⁹-THC are pooled, thereby producing an extract with Δ⁹-THC substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged (for example, at least 2, 3, 4, 5, 6, 7 or 8 or more, or between 3 and 30, columns are arranged) in a continuous rotation traveling through a series of contact points where gradient elution solvents s and extract solutions are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution solvent is introduced (or loaded) while at the same time a second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced and a third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent solution is introduced, the second column moves to the fifth position where the fourth gradient solvent is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced.

In an alternative embodiment, the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent.

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged, for example 3, 4, 5, 6, 7, 8 or more, or between 3 and 30 or more, columns or more are arranged in a continuous rotation traveling through a series of contact points where gradient elution solvents s and extract solutions are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution solvent is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent i is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent is introduced, the second column moves to the fifth position where the fourth gradient solvent is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent solution is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced. The equipment is generally described in U.S. Pat. Nos. 4,764,276, 4,808,317, and 4,710,364, for example, each of which are incorporated herein by reference.

In a particular embodiment the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient solvent elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient solvent elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent. The second gradient solvent containing CBD is combined with a Lewis acid or non-oxidizing acid catalyst, optionally additional CBD or substantially pure CBD and/or (Δ8-THC) and/or Δ9-tetrahydrocannabinol (Δ9-THC) can be added to the reaction mixture, mixing said reaction mixture for a period of time; adding a neutralizing agent to said mixture; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally dissolving organic phase in a second solvent; adding a stabilizing agent; mixing said reaction mixture for a period of time; adding a elimination catalyst or reagent to said reaction mixture; filtration of stabilizing agent and elimination agent from mixture; optionally allowing mixture to separate into an aqueous phase and an organic phase; optionally adding a further solvent; adding organic phase to a chromatography column and eluting the tetrahydrocannabinol from the organic phase.

The tetrahydrocannabinol may then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase).

In some embodiments, (substantially) only CBD is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of CBD. These fractions free of CBD are pooled, thereby producing an extract with CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, Δ⁹-THC carboxylic acid species or Δ⁹-THCA and CBD carboxylic acid species CBDA are fractionated out of out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a first elution are passed over the column, Δ⁹-THCA and CBDA are differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled first elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THCA and CBDA. These fractions (substantially) free of Δ⁹-THCA and CBDA are pooled, thereby producing an extract with Δ⁹-THCA and CBDA substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, the Δ⁹-THC may be eluted from the column, extracted or concentrated, for purifying, or substantially purifying, Δ⁹-THC. In alternative embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) a specific cannabinoid or groups of cannabinoids or their carboxylic acid species, cannflavin or essential oil or class of cannabinoids, cannflavins or essential oils out of the eluent, for example, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabidivarol (CBDV), tetrahydrocannabidiol (THCBD), tetrahydrocannabigerol (THCBG), tetrahydrocannabichromene (THCBC), tetrahydrocannabidivarol (THCBDV), Δ⁸-THC, the carboxylic acid precursors of the foregoing compounds, and related naturally occurring compounds and their derivatives. In alternate embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) cannflavins and related naturally occurring compounds and their derivatives. In alternate embodiments, the system is arranged to fractionate the components of essential oils. The list of compounds provided herein is not exhaustive and is in no way intended to be limiting. In these embodiments, the compound(s) of interest are retained or detained (e.g., reversibly bound) on the column so that fractions (alternatively, the last fractions) of the extract eluted from the column contain the compounds(s) of interest. In alternative embodiments, fractions containing the compound(s) of interest are pooled. In some embodiments, different compounds may be extracted with different solvents and then combined into a single extract. As will be appreciated by one knowledgeable in the art, in this manner, several different cannabinoids could be purified from a single extract.

In alternative embodiments, the “plant or microbial material” will be derived from one or more Cannabis or hemp plants, or from other plants, or microbes such as lichen, yeast, algae or bacteria. The term “plant material” encompasses a plant or plant part (e.g. bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries or parts thereof) as well as exudates, and includes material falling within the definition of “botanical raw material” in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research. The term “Cannabis plant(s)” encompasses wild type Cannabis sativa and also variants thereof, including Cannabis chemovars (varieties characterized by virtue of chemical composition) which naturally contain different amounts of the individual cannabinoids, also Cannabis sativa subspecies indica including the variants var. indica and var. kafiristanica, Cannabis indica and also plants which are the result of genetic crosses, self-crosses or hybrids thereof. The term “Cannabis plant material” also can encompass plant material derived from one or more Cannabis plants, and can comprise any “Cannabis plant material” including, e.g., herbal Cannabis and dried Cannabis biomass. The term “Cannabis plant material” also can encompass “decarboxylated Cannabis plant material”, which refers to Cannabis plant material which has been subject to a decarboxylation step in order to convert cannabinoid acids to the corresponding free cannabinoids.

In alternative embodiments, a starting material for a purification process as provided herein is an extract solution containing a cannabinoid or cannabinoid acid obtained from a natural or a synthetic source, e.g., a plant or microbial material. In alternative embodiments, the “extract solution containing a cannabinoid or cannabinoid acid” comprises a solvent extract of a plant or microbial material. Solvents used for extraction for use in the preparation of extract solutions can comprise non-polar solvents, polar solvents such as ethanol, methanol or water, or organic solvents such as liquid carbon dioxide, and combinations thereof. The solvent may be an organic solvent, selected from the group consisting of: non-polar solvents include liquid non-polar solvents comprising lower C1-C12, preferably C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol, methanol, water; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; or a supercritical fluid such as CO₂ with or without an organic solvent modifier.

In alternative embodiments, the extract is prepared by dissolving or partially dissolving the natural or synthetic, or the plant or microbial material in a solvent, removing insoluble material from the resultant solution (optionally by filtration with or without activated carbon, precipitation, centrifugation and the like), and optionally removing some or all of the extraction solvent from the solution (optionally by rotary evaporation) to form an extract or extract solution or concentrate containing a cannabinoid or cannabinoid acid.

In alternative embodiments, extractions can comprise using a technique referred to as accelerated solvent extraction or may use subcritical water or any combination of water and solvent. In one embodiment, when isolating cannabinoid acids, a modified pH gradient elution solvent is used. The primary purpose of this pH adjustment (the modified pH gradient) is to promote or prevent ionization of the cannabinoid acid. pH modified gradient elution solutions may be achieved by the additional of a small volume of acid or base to the solvent. It may be sufficient to add a relatively weak acid, such as acetic acid, oxalic acid, glycolic acid, carbonic acid or ammonium hydroxide or a small amount of base or buffering agent such as sodium hydroxide, magnesium hydroxide, sodium carbonate or sodium bicarbonate. For any given purification process the optimal amount and type of acid or base used may be determined empirically. An alternative exemplary acidified solvent is 0.1% acetic acid in ethanol or 0.1% sodium hydroxide in ethanol. In alternative embodiments, the neutralizing agent consists of for example sodium hydroxide, sodium carbonate, potassium carbonate, and potassium t-amylate, sodium bicarbonate.

Acidified non-polar and polar solvents of the types described above can be useful in preparation of gradient elutions using ion exchange chromatography. The solvents used in the conversion can comprise an organic solvent, e.g., a non-polar solvent, including a liquid non-polar solvent comprising lower C1-C12, or C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane, hexane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; a heterocyclic compound or cyclic ether for example, tetrahydrofuran and 2-Methyltetrahydrofuran and aromatic ring hydrocarbons such as benzene, toluene, xylene and ethylbenzene.

In alternative embodiments, the elimination catalyst consists of a catalyst and or reagent such as, for example, lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)₂), calcium hydroxide (Ca(OH)₂), strontium hydroxide (Sr(OH)₂), barium hydroxide (Ba(OH)₂), potassium hydride and/or sodium hydride, potassium tert-pentoxide, organic superbases, bispidines, multicyclic polyamines, organometallic compounds of reactive metals, wherein optionally the reactive metals comprise organolithium, organo-magnesium, lithium diisopropylamide, n-butyllithium and potassium tert-butoxide, potassium tert-pentoxide, lithium nitride, sodium methoxide, sodium ethoxide, ortho-diethynylbenzene dianion, meta-diethynylbenzene dianion, para-diethynylbenzene dianion, lithium monoxide anion, and/or a methyl anion.

In alternative embodiments, the plant or microbial material is subjected to a decarboxylation step prior to solvent extraction. The purpose of the decarboxylation step is to convert cannabinoid acids present in the plant or microbial material to the corresponding free cannabinoids. In alternative embodiments, the decarboxylation is carried out by heating the plant or microbial material to a defined temperature for a suitable length of time. Decarboxylation of cannabinoid acids is a function of time and temperature, thus at higher temperatures a shorter period of time will be taken for complete decarboxylation of a given amount of cannabinoid acid. In alternative embodiments selecting appropriate conditions for decarboxylation consideration includes minimizing thermal degradation of the desirable, pharmacological cannabinoids into undesirable degradation products, particularly thermal degradation of Δ⁹-THC. In alternative embodiments, the decarboxylation is carried out in a multi-step heating process in which the plant or microbial material is: i) heated to a first temperature for a first (relatively short) time period to evaporate off retained water and allow for uniform heating of the plant or microbial material; and ii) the temperature is increased to a second temperature for a second time period (typically longer than the first time period) until at least 95% conversion of the acid cannabinoids to their neutral form has occurred.

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” prepared from the starting plant or microbial material comprises a “botanical drug substance” prepared from the plant or microbial material, or a polar or non-polar solvent solution of such a botanical drug substance. In the context of this application a “botanical drug substance” is an extract derived from plant or microbial material, which extract fulfills the definition of “botanical drug substance” provided in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research of: “A drug substance derived from one or more plants, algae, or macroscopic fungi. It is prepared from botanical raw materials by one or more of the following processes: pulverization, decoction, expression, aqueous extraction, ethanolic extraction, or other similar processes.” “Botanical drug substances” derived from Cannabis plants include primary extracts prepared by such processes as, for example, maceration, percolation, and solvent extraction.

In alternative embodiments, solvent extraction may be carried out using essentially any solvent that dissolves, or substantially dissolves, cannabinoids/cannabinoid acids, such as for example C1 to C5 alcohols (e.g. ethanol, methanol), C5-C12 alkanes (e.g. hexane), norflurane (HFA134a), 1,1,1,2,3,3,3-Heptafluoropropane (or HFA227), chloroform, dichloromethane, dichloroethane and carbon dioxide. When solvents such as those listed above are used, the resultant extract typically contains non-specific lipid-soluble material. This can optionally be removed by a variety of processes including filtration to remove solids, “winterization”, which involves for example chilling to −20° C. or lower followed by filtration to remove waxy ballast, extraction with liquid carbon dioxide and by distillation.

In alternative embodiments, any protocol for the preparation of botanical drug substances from Cannabis and hemp plant material can be used, e.g., as described in International patent application WO 02/064109. In alternative embodiments, the botanical drug substance is obtained by carbon dioxide (CO₂) extraction, polar solvent extraction or non-polar solvent extraction or combinations thereof followed by a filtration. Optionally a secondary extraction is performed to remove a substantial proportion of non-cannabinoid materials, e.g. waxes, wax esters and glycerides, unsaturated fatty acid residues, terpenes, carotenes, and flavonoids and other ballast.

In alternative embodiments, if it is intended to prepare free cannabinoids from the plant or microbial material, e.g., Cannabis, then the material is heated to a defined temperature for a defined period of time in order to partially or substantially decarboxylate cannabinoid acids to free cannabinoids prior to extraction of the botanical drug substance. In alternative embodiments, the botanical drug substance is prepared according to a process comprising the following steps: i) optional decarboxylation of the plant or microbial material, ii) extraction with polar or non-polar solvent, to produce a crude botanical drug substance, iii) optional precipitation with C1-C5 alcohol to reduce the proportion of non-target materials, iv) removal of the precipitate (preferably by filtration), v) optional treatment with activated charcoal, and vi) evaporation to remove C1-C5 alcohol and water, thereby producing a final botanical drug substance.

In alternative embodiments, provided are methods for converting first substantially purified cannabidiol (CBD) to Δ⁸-tetrahydrocannabinol (Δ⁸-THC) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC), and then further converting the produced Δ9-tetrahydrocannabinol (Δ⁹-THC) to Δ⁸-tetrahydrocannabinol (Δ⁸-THC). As will be appreciated by one knowledgeable in the art and as discussed below, the reaction times may be varied somewhat, producing product at different yields and purities. Furthermore, functional equivalents may be substituted where appropriate.

In alternative embodiments, an exemplary method of converting CBD to Δ⁸-tetrahydrocannabinol (Δ⁸-THC) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and further converting (Δ⁹-THC) to (Δ⁸-THC) comprises: providing a reaction mixture comprising a Lewis acid or non-oxidizing acid catalyst in a reaction solvent, adding a substantially pure CBD or substantially pure CBD and/or (Δ⁸-THC) and/or Δ⁹-tetrahydrocannabinol (Δ⁹-THC) to the reaction mixture, mixing said reaction mixture for a period of time; adding a neutralizing agent to said mixture; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally dissolving organic phase in a second solvent; adding a stabilizing agent; mixing said reaction mixture for a period of time; adding a elimination catalyst or reagent to said reaction mixture; filtration of stabilizing agent and elimination agent from mixture; optionally allowing mixture to separate into an aqueous phase and an organic phase; optionally adding a further solvent; adding organic phase to a chromatography column and eluting the tetrahydrocannabinol from the organic phase. The tetrahydrocannabinol may then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase.

In alternative embodiments, the tetrahydrocannabinol at therapeutically effective concentrations or dosages is combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, methacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients that can be used include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethyl cellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethycellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example, Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed.

In some embodiments, the catalyst is a Lewis acid, for example, p-toluenesulfonic acid, boron trifluoride or BF₃Et₂O. In some embodiments, the BF₃Et₂O (boron trifluoride diethyl etherate) is in dry methylene chloride, ethylethyl acetate, ethanol, hexane or other organic solvent. In yet other examples, the catalyst may be hydrochloric acid in ethanol or sulfuric acid in cyclohexane.

In some embodiments, the catalyst is a non-oxidizing acid, for example, formic acid, acetic acid or hydrobromic acid. In some embodiments, the non-oxidizing acid is in dry methylene chloride, ethyl acetate, ethanol, hexane or other organic solvent.

In some embodiments, a base is added to the reaction mixture prior to allowing the reaction mixture to separate into organic and aqueous phases. The base may be an alkali metal hydrogen carbonate, carbonate of an alkali metal, lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)), calcium hydroxide (Ca(OH)), strontium hydroxide (Sr(OH)), barium hydroxide (Ba(OH)).

In some embodiments, the organic layer is dried prior to eluting. In these embodiments, a suitable drying or dehydration compound, for example, MgSO4 or Na₂SO₄ is used.

In yet other embodiments, the process may be carried out under a nitrogen (e.g., N₂) atmosphere.

In alternative embodiments, and as discussed below, yield is determined by looking at the peak area for the isolated compound in the gas chromatography-mass spectra analysis of the crude reaction product mixture and the final reaction product mixture. It is important to note that in the prior art, yield is often calculated on the basis of first isolated crude product before final purification. In some embodiments of processes provided herein yield of Δ9-THC is at least about 75%; in other embodiments, the yield of Δ9-THC is at least about 90%; and in other embodiments, yield of Δ9-THC is at least about 98%; and in yet other embodiments, yield of Δ9-THC is between about 75 to 98% or 99%.

In alternative embodiments, purity is determined by Gas Chromatography-Mass Spectrometry (GC-MS) and/or by analytical high-performance liquid chromatography (HPLC). The total ion chromatogram from the GC-MS gives information similar to that provided by a flame ionization detector (FID)-GC in that the peak area is proportional to the mass of the analytes detected. Total peak area and the peak areas of the individual analytes can be compared in the GC-MS case as long as the masses are in generally the same range. As discussed below, in some embodiments, purity of the Δ9-THC isolated by the process is greater than about 70%, 80%, 90%, 95%, 97% or 98% or more, or purity is greater that about 98% to 99% or 90% to 99.5%.

Chapter III

In alternative embodiments, provided are continuous isolation and purification processes for preparing a substantially pure cannabidiol or a product enriched in cannabidiol from plant or microbial material extracts. In alternative embodiments, provided herein are improved methods for converting cannabidiol (CBD) to Δ⁸-THC, including a purification and conversion process based on a simple combination of continuous chromato-graphic gradient elutions and semi continuous isomerization reactions. This exemplary process is simple, efficient and economic.

In alternative embodiments, provided are methods of continuous isolation of cannabinoids in substantially pure form starting from plant extract material and conversion of the purified CBD to form both Δ⁸ THC and Δ⁹ THC and subsequent conversion of the formed Δ⁹ tetrahydrocannabinol into Δ⁸ tetrahydrocannabinol.

In alternative embodiments, provided are processes for producing and isolating cannabinoids from Cannabis and hemp extracts which contain cannabinoids in minute amounts. In alternative embodiments, provided are processes for producing and isolating cannabinoids from natural materials, including plant or plant extracts, microbes, or botanical drug substances, or synthetically and semi-synthetically prepared cannabinoid products, or from recombinantly engineered microbes, e.g., yeasts or bacteria recombinantly engineered to express one or more cannabinoids. In one embodiment, exemplary methods are inexpensive and provide specific cannabinoid concentrates (e.g., of CBD, Δ⁸-THC, Δ⁹-THC) of high purity.

In one embodiment, exemplary methods provide a simple and economical continuous process for separating and concentrating cannabinoids from solvent-extracted cannabinoid containing materials. In one embodiment, exemplary methods provide a method that first converts the substantially isolated CBD into a mixture of Δ⁸-THC and Δ⁹-THC, and then subsequently converts the Δ⁹-THC into Δ⁸-THC. In alternative embodiments, the solvent-extracted cannabinoid containing materials are derived from synthetic or biological materials such as hemp and Cannabis or botanical drug substances, or from microbial materials; and the solvent extraction methods can be polar solvent extractions, nonpolar solvent extractions, or the solvent extraction methods can comprise use of super critical carbon dioxide or mixtures thereof. The solvent extraction methods can extract cannabinoids substantially from the synthetic or biological, e.g., plant, matter, along with other plant matter comprising lipids, waxes, monoterpenes, sesquiterpenes, hydrocarbons, alkaloids, flavonoids and chlorophylls.

In alternative embodiments, methods provided herein comprise subjecting cannabinoid containing solvent extract starting materials to a number of chromatographic resins in various contacting steps using various gradient elution solutions.

In alternative embodiments, the cannabinoids which can be fractionated and isolated using methods as provided herein, or which can be produced in reactions as provided herein, or from which the solvent extracts are derived can be from, or can comprise: Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetrahydrocannabidiol (THCBD); tetrahydrocannabigerol (THCBG); tetrahydrocannabichromene (THCBC); tetrahydrocannabidivarol (THCBDV), or combinations thereof, including carboxylic acid precursors of the foregoing compounds and related naturally occurring compounds and their derivatives.

In alternative embodiments, provides are methods of preparing or obtaining a substantially pure cannabinoid or a product enriched in a given cannabinoid comprising:

-   -   (i) obtaining an extract or extract solution containing a         cannabinoid or a cannabinoid acid from a plant or microbial         material;     -   (ii) filtering the extract of step (i) to remove solids and         color bodies;     -   (iii) removing the extract solvent (as an extract fraction);     -   (v) continuously loading of an amount of extract solution over a         defined time increment over multiple stationary phase resins         columns such as a normal phase, reverse phase and/or ion         exchange chromatographic resin;     -   (vi) continuously eluting the extract solution using multiple         defined gradient elution solutions at specific time increments         and volumes;     -   (vii) continuously collecting the gradient elution fractions;     -   (viii) removal of the gradient elution solvent from the produced         fractions to generate a substantially purified extract     -   (ix) optionally loading of specific first gradient elution         fractions on a reverse phase, ion exchange or normal         chromatographic resin;     -   (x) continuously eluting the first gradient elution fractions         with a second gradient elution solvent; and,     -   (xi) removing of the second gradient elution solvent from the         produced fractions to produce a purified extract.

In alternative embodiments, the methods further comprise:

-   -   a step (xii): loading of first and second gradient elution         solvents s onto an ion exchange chromatographic resin;     -   a step (xiii): eluting an extract solution from the ion exchange         chromatographic resin of step (xi) using a gradient solvent and         collecting the gradient elution fractions; and     -   (xiv) removing the gradient elution solution from the produced         fractions of step (xiii) to produce purified and substantially         purified extract.

In alternative embodiments, provided are methods of converting CBD to Δ⁸THC and Δ⁹ THC comprising:

-   -   (a) providing a reaction mixture comprising a catalyst in an         organic solvent;     -   (b) adding CBD material from steps (viii) and steps (xiii),         above;     -   (c) mixing said reaction mixture;     -   (d) allowing the mixture to separate into an aqueous phase and         an organic phase;     -   optionally adding a second solvent; and     -   (e) removing the organic phase; and,     -   optionally, repeating steps (a) through (e).

In alternative embodiments, provided are methods for converting CBD to substantially pure Δ⁸ THC comprising:

-   -   (a) providing a reaction mixture comprising a CBD in an organic         solvent;     -   (b) adding a catalyst to the reaction mixture under a nitrogen         atmosphere;     -   (c) stirring the reaction mixture;     -   (d) controlling the reaction temperature and continuously         measuring the rate of reaction by observing conversion of CBD to         first Δ⁹-THC and second order of reaction Δ⁸-THC;     -   (d) adding NaHCO₃ or equivalent to the reaction mixture once         Δ⁹-THC has be substantially converted to Δ⁸-THC;     -   (e) allowing the mixture to separate into an aqueous phase and         an organic phase and optionally adding a second solvent;     -   (f) removing the organic phase, which comprises substantially         the converted Δ⁸-THC; and     -   optionally repeating steps (a) through (f);     -   optionally performing steps (v) through (xiv).

In alternative embodiments, a “substantially pure” preparation of cannabinoid is defined as a preparation having a chromatographic purity (of the desired cannabinoid or cannabinoid acid) of greater than about 75%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99%, or greater than about 99.5%, or between about 70% and 99.9%, as determined by area normalisation of an HPLC profile.

In alternative embodiments, the term “product enriched in a given cannabinoid” encompasses preparations having at least about 50%, or greater than about 75%, or greater than about 90%, 95% or 98%, or between about 50% and 99.9%, chromatographic purity, for the desired cannabinoid.

In alternative embodiments, a non-purified, or non-substantially purified, product can comprise a greater proportion of impurities, non-target materials and/or other cannabinoids than a “substantially pure” preparation. The cannabinoid can be (e.g., a cannabinoid purified or isolated by, or made by a reaction of, a method as provided herein can be): Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetra-hydrocannabidiol (THCBD); tetra-hydrocannabigerol (THCBG); tetra-hydrocannabichromene (THCBC); or, tetra-hydrocannabidivarol (THCBDV); the carboxylic acid precursors of the foregoing compounds; and related naturally occurring compounds and their derivatives.

In alternative embodiments, the term “cannabinoids” e.g., a cannabinoid purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,1′-di-methyl-pyrane ring, a variedly derivatized aromatic ring and/or a variedly unsaturated cyclohexyl ring and their immediate chemical precursors.

In alternative embodiments, the term “cannflavins”, e.g., a cannflavin purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,4-pyrone ring fused to a variedly derivatized aromatic ring and linked to a second variedly derivatized aromatic ring.

In alternative embodiments, the term “Lewis acid” refers to a powerful electron pair acceptor; and examples include but are by no means limited to BF₃Et₂O (boron trifluoride ditheyl etherate), p-toluenesulfonic acid and boron trifluoride.

In alternative embodiments, the term “non-oxidizing acid” refers to hydrobromic, hydrochloric, hydrofluoric, acetic, benzoic, chloroacetic, formic, phosphoric, sulfuric, trifluroacetic and oxalic acids.

In alternative embodiments, the term “stabilizing agent” refers to a zinc chloride, hydrogen chloride, methylene chloride, simonkolleite, and mixtures thereof; and examples include but are by no means limited to zinc chloride, hydrogen chloride, simonkolleite, and polymorphs and hydrates thereof.

In alternative embodiments, the term “essential oils”, e.g., an essential oil used as a starting material in a process as provided herein, or an essential oil that may be isolated by a process as provided herein, includes or refers to a family of natural products that can contain a multiple of the 5-membered isoprene unit variedly substituted, often cyclized to form one or more ring systems; they may also contain series of aldehydes and/or ketones and esters of a variety of carboxylic acid substituted compounds.

In alternative embodiments, provided are methods for extracting and/or purifying cannabinoids from any plant or microbial material extract known to contain such cannabinoids, cannflavins and essential oils; and, optionally to purify cannflavins and to optionally purify essential oils. In alternative embodiments, the extract is passed through a series of chromatographic columns, for example, a normal phase column, a reversed phase column or an ion exchange column as a continuous column configuration.

In one embodiment, the chromatographic column is arranged for gradient elution fractioning using normal phase, reverse phase and/or ion exchange chromatography. In one embodiment Δ⁹-THC and Δ⁸-THC and CBD are fractionated out of the eluent. For example, in one embodiment, as the extract is passed over the column, Δ⁹-THC, Δ⁸-THC and CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁸-THC and CBD. The fractions free of Δ⁸-THC and CBD are pooled, thereby producing an extract with Δ⁹-THC, Δ⁸-THC and CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, (substantially) only Δ⁸-THC is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁸-THC is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extracts are pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be free of Δ⁸-THC. These fractions free of Δ⁸-THC are pooled, thereby producing an extract with Δ⁹-THC substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged, for example 3, 4, 5, 6, 7, 8 or more, or between 3 and 30 or more, columns are arranged in a continuous rotation traveling through a series of contact points where gradient elution solutions and extract solution are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent is introduced, the second column moves to the fifth position where the fourth gradient solvent is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced. The equipment is generally described in U.S. Pat. Nos. 4,764,276, 4,808,317, and 4,710,364, for example, each of which are incorporated herein by reference.

In a particular embodiment the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent.

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged for example 3, 4, 5, 6, 7, 8 or more, or between 3 and 30 or more, columns are arranged in a continuous rotation traveling through a series of contact points where gradient elution solutions and extract solution are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent solution is introduced, the second column moves to the second position where the first gradient solvent is introduced and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent is introduced, the second column moves to the fifth position where the fourth gradient solvent is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced. In a particular embodiment the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent. The second gradient solvent containing CBD is combined with a Lewis acid or non-oxidizing acid catalyst, optionally additional CBD or substantially pure CBD and/or Δ9-tetrahydrocannabinol (Δ9-THC) can be added to the reaction mixture, mixing said reaction mixture for a period of time at a controlled temperature to first convert CBD to Δ⁹-THC and continue to the second order reaction to convert formed Δ⁹-THC to second order reaction Δ⁸-THC; adding a neutralizing agent to said mixture once substantial conversion of Δ⁹-THC to Δ⁸-THC is observed by sample analysis such as TLC, HPLC or GCMS; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally adding a further solvent; adding organic phase to a continuous or fixed chromatography column and eluting the Δ⁸-THC tetrahydrocannabinol from the organic phase. The tetrahydrocannabinol may then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase.

In some embodiments, (substantially) only CBD is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of CBD. These fractions free of CBD are pooled, thereby producing an extract with CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, Δ⁹-THC carboxylic acid species or Δ⁹-THCA and CBD carboxylic acid species CBDA are fractionated out of out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a first elution are passed over the column, Δ⁹-THCA and CBDA are differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled first elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THCA and CBDA. These fractions (substantially) free of Δ⁹-THCA and CBDA are pooled, thereby producing an extract with Δ⁹-THCA and CBDA substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, the Δ⁹-THC may be eluted from the column, extracted or concentrated, for purifying, or substantially purifying, Δ⁹-THC. In alternative embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) a specific cannabinoid or groups of cannabinoids or their carboxylic acid species, cannflavin or essential oil or class of cannabinoids, cannflavins or essential oils out of the eluent, for example, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabidivarol (CBDV), tetrahydrocannabidiol (THCBD), tetrahydrocannabigerol (THCBG), tetrahydrocannabichromene (THCBC), tetrahydrocannabidivarol (THCBDV), Δ⁸-THC, the carboxylic acid precursors of the foregoing compounds, and related naturally occurring compounds and their derivatives. In alternate embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) cannflavins and related naturally occurring compounds and their derivatives. In alternate embodiments, the system is arranged to fractionate the components of essential oils. The list of compounds provided herein is not exhaustive and is in no way intended to be limiting. In these embodiments, the compound(s) of interest are retained or detained (e.g., reversibly bound) on the column so that fractions (alternatively, the last fractions) of the extract eluted from the column contain the compounds(s) of interest. In alternative embodiments, fractions containing the compound(s) of interest are pooled. In some embodiments, different compounds may be extracted with different solvents and then combined into a single extract. As will be appreciated by one knowledgeable in the art, in this manner, several different cannabinoids could be purified from a single extract.

In alternative embodiments, the “plant material or microbial” will be derived from one or more Cannabis or hemp plants, or from other plants, or microbes such as yeast, lichen, algae or bacteria. The term “plant or microbial material” encompasses a plant or plant part (e.g. bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries or parts thereof) as well as exudates, and includes material falling within the definition of “botanical raw material” in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research. The term “Cannabis plant(s)” encompasses wild type Cannabis sativa and also variants thereof, including Cannabis chemovars (varieties characterized by virtue of chemical composition) which naturally contain different amounts of the individual cannabinoids, also Cannabis sativa subspecies indica including the variants var. indica and var. kafiristanica, Cannabis indica and also plants which are the result of genetic crosses, self-crosses or hybrids thereof. The term “Cannabis plant material” also can encompass plant material derived from one or more Cannabis plants, and can comprise any “Cannabis plant material” including, e.g., herbal Cannabis and dried Cannabis biomass. The term “Cannabis plant material” also can encompass “decarboxylated Cannabis plant material”, which refers to Cannabis plant material which has been subject to a decarboxylation step in order to convert cannabinoid acids to the corresponding free cannabinoids.

In alternative embodiments, a starting material for a purification process as provided herein is an extract solution containing a cannabinoid or cannabinoid acid obtained from a natural or a synthetic source, e.g., a plant or microbial material. In alternative embodiments, the “extract solution containing a cannabinoid or cannabinoid acid” comprises a solvent extract of a plant or microbial material. Solvents used for extraction for use in the preparation of extract solutions can comprise non-polar solvents, polar solvents such as ethanol, methanol or water, or organic solvents such as liquid carbon dioxide, and combinations thereof. The solvent may be an organic solvent, selected from the group consisting of: non-polar solvents include liquid non-polar solvents comprising lower C1-C12, preferably C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol, methanol, water; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; or a supercritical fluid such as CO₂ with or without an organic solvent modifier.

In alternative embodiments, the extract is prepared by dissolving or partially dissolving the natural or synthetic, or the plant or microbial material in a solvent, removing insoluble material from the resultant solution (optionally by filtration with or without activated carbon, precipitation, centrifugation and the like), and optionally removing some or all of the extraction solvent from the solution (optionally by rotary evaporation) to form an extract or extract solution or concentrate containing a cannabinoid or cannabinoid acid.

In alternative embodiments, extractions can comprise using a technique referred to as accelerated solvent extraction or may use subcritical water or any combination of water and solvent. In one embodiment, when isolating cannabinoid acids, a modified pH gradient elution solution is used. The primary purpose of this pH adjustment (the modified pH gradient) is to promote or prevent ionization of the cannabinoid acid. pH modified gradient elution solutions may be achieved by the additional of a small volume of acid or base to the solvent. It may be sufficient to add a relatively weak acid, such as acetic acid, oxalic acid, glycolic acid, carbonic acid or ammonium hydroxide or a small amount of base or buffering agent such as sodium hydroxide, magnesium hydroxide, sodium carbonate or sodium bicarbonate. For any given purification process the optimal amount and type of acid or base used may be determined empirically. An alternative exemplary acidified solvent is 0.1% acetic acid in ethanol or 0.1% sodium hydroxide in ethanol. In alternative embodiments, the neutralizing agent consists of for example sodium hydroxide, sodium carbonate, potassium carbonate, and potassium t-amylate, sodium bicarbonate.

Acidified non-polar and polar solvents of the types described above can be useful in preparation of gradient elutions using ion exchange chromatography. The solvents used in the conversion can comprise an organic solvent, e.g., a non-polar solvent, including a liquid non-polar solvent comprising lower C1-C12, or C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane, hexane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; a heterocyclic compound or cyclic ether for example, tetrahydrofuran and 2-Methyltetrahydrofuran and aromatic ring hydrocarbons such as benzene, toluene, xylene and ethylbenzene.

In alternative embodiments, the plant or microbial material is subjected to a decarboxylation step prior to solvent extraction. The purpose of the decarboxylation step is to convert cannabinoid acids present in the plant or microbial material to the corresponding free cannabinoids. In alternative embodiments, the decarboxylation is carried out by heating the plant or microbial material to a defined temperature for a suitable length of time. Decarboxylation of cannabinoid acids is a function of time and temperature, thus at higher temperatures a shorter period of time will be taken for complete decarboxylation of a given amount of cannabinoid acid. In alternative embodiments selecting appropriate conditions for decarboxylation consideration includes minimizing thermal degradation of the desirable, pharmacological cannabinoids into undesirable degradation products, particularly thermal degradation of Δ⁹-THC. In alternative embodiments, the decarboxylation is carried out in a multi-step heating process in which the plant or microbial material is: i) heated to a first temperature for a first (relatively short) time period to evaporate off retained water and allow for uniform heating of the plant or microbial material; and ii) the temperature is increased to a second temperature for a second time period (typically longer than the first time period) until at least 95% conversion of the acid cannabinoids to their neutral form has occurred.

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” prepared from the starting plant or microbial material comprises a “botanical drug substance” prepared from the plant material, or a polar or non-polar solvent solution of such a botanical drug substance. In the context of this application a “botanical drug substance” is an extract derived from plant or microbial material, which extract fulfills the definition of “botanical drug substance” provided in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research of: “A drug substance derived from one or more plants, algae, or macroscopic fungi. It is prepared from botanical raw materials by one or more of the following processes: pulverization, decoction, expression, aqueous extraction, ethanolic extraction, or other similar processes.” “Botanical drug substances” derived from Cannabis plants include primary extracts prepared by such processes as, for example, maceration, percolation, and solvent extraction.

In alternative embodiments, solvent extraction may be carried out using essentially any solvent that dissolves, or substantially dissolves, cannabinoids/cannabinoid acids, such as for example C1 to C5 alcohols (e.g. ethanol, methanol), C5-C12 alkanes (e.g. hexane), norflurane (HFA134a), 1,1,1,2,3,3,3-Heptafluoropropane (or HFA227), chloroform, dichloromethane, dichloroethane and carbon dioxide. When solvents such as those listed above are used, the resultant extract typically contains non-specific lipid-soluble material. This can optionally be removed by a variety of processes including filtration to remove solids, “winterization”, which involves for example chilling to −20° C. or lower followed by filtration to remove waxy ballast, extraction with liquid carbon dioxide and by distillation.

In alternative embodiments, any protocol for the preparation of botanical drug substances from Cannabis and hemp plant material can be used, e.g., as described in International patent application WO 02/064109. In alternative embodiments, the botanical drug substance is obtained by carbon dioxide (CO₂) extraction, polar solvent extraction or non-polar solvent extraction or combinations thereof followed by a filtration. Optionally a secondary extraction is performed to remove a substantial proportion of non-cannabinoid materials, e.g. waxes, wax esters and glycerides, unsaturated fatty acid residues, terpenes, carotenes, and flavonoids and other ballast.

In alternative embodiments, if it is intended to prepare free cannabinoids from the plant or microbial material, e.g., Cannabis, then the material is heated to a defined temperature for a defined period of time in order to partially or substantially decarboxylate cannabinoid acids to free cannabinoids prior to extraction of the botanical drug substance. In alternative embodiments, the botanical drug substance is prepared according to a process comprising the following steps: i) optional decarboxylation of the plant or microbial material, ii) extraction with polar or non-polar solvent, to produce a crude botanical drug substance, iii) optional precipitation with C1-C5 alcohol to reduce the proportion of non-target materials, iv) removal of the precipitate (preferably by filtration), v) optional treatment with activated charcoal, and vi) evaporation to remove C1-C5 alcohol and water, thereby producing a final botanical drug substance.

In alternative embodiments, provided are methods for converting first substantially purified cannabidiol (CBD) to Δ8-tetrahydrocannabinol (Δ8-THC) and Δ9-tetrahydrocannabinol (Δ9-THC), and then further converting the produced Δ9-tetrahydrocannabinol (Δ9-THC) to Δ8-tetrahydrocannabinol (Δ8-THC). As will be appreciated by one knowledgeable in the art and as discussed below, the reaction times may be varied somewhat, producing product at different yields and purities. Furthermore, functional equivalents may be substituted where appropriate.

In alternative embodiments, an exemplary method of converting CBD to Δ8-tetrahydrocannabinol (Δ8-THC) and Δ9-tetrahydrocannabinol (Δ9-THC) and further converting (Δ9-THC) to (Δ8-THC) comprises: providing a reaction mixture comprising a Lewis acid or non-oxidizing acid catalyst in a reaction solvent, adding a substantially pure CBD or substantially pure CBD and/or (Δ9-THC) to the reaction mixture, mixing said reaction mixture for a period of time; adding a neutralizing agent to said mixture when it is observed that Δ8-tetrahydrocannabinol (Δ8-THC) is substantially formed and Δ9-tetrahydrocannabinol (Δ9-THC) has been substantially eliminated; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally adding a further solvent; adding organic phase to a continuous or fixed bed chromatography column and eluting the Δ8-tetrahydrocannabinol (Δ8-THC) from the organic phase. The tetrahydrocannabinol may then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase).

In alternative embodiments, the tetrahydrocannabinol at therapeutically effective concentrations or dosages is combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, methacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients that can be used include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethyl cellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethycellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example, Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed.

In some embodiments, the catalyst is a Lewis acid, for example, p-toluenesulfonic acid, boron trifluoride or BF₃Et₂O. In some embodiments, the BF₃Et₂O (boron trifluoride diethyl etherate) is in dry methylene chloride, ethyl acetate, ethanol, hexane or other organic solvent. In yet other examples, the catalyst may be hydrochloric acid in ethanol or sulfuric acid in cyclohexane.

In some embodiments, the catalyst is a non-oxidizing acid, for example, formic acid, acetic acid or hydrobromic acid. In some embodiments, the non-oxidizing acid is in dry methylene chloride, ethyl acetate, ethanol, hexane or other organic solvent.

In some embodiments, a base is added to the reaction mixture prior to allowing the reaction mixture to separate into organic and aqueous phases. The base may be an alkali metal hydrogen carbonate, carbonate of an alkali metal, lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)), calcium hydroxide (Ca(OH)), strontium hydroxide (Sr(OH)), barium hydroxide (Ba(OH)).

In some embodiments, the organic layer is dried prior to eluting. In these embodiments, a suitable drying or dehydration compound, for example, MgSO4 or Na₂SO₄ is used.

In yet other embodiments, the process may be carried out under a nitrogen (e.g., N₂) atmosphere.

In alternative embodiments, and as discussed below, yield is determined by looking at the peak area for the isolated compound in the gas chromatography—mass spectra analysis of the crude reaction product mixture and the final reaction product mixture. It is important to note that in the prior art, yield is often calculated on the basis of first isolated crude product before final purification. In some embodiments of processes provided herein yield of Δ8-THC is at least about 75%; in other embodiments, the yield of Δ8-THC is at least about 90%; and in other embodiments, yield of Δ8-THC is at least about 98%; and in yet other embodiments, yield of Δ8-THC is between about 75 to 98% or 99%.

In alternative embodiments, purity is determined by Gas Chromatography-Mass Spectrometry (GC-MS) and/or by analytical high-performance liquid chromatography (HPLC). The total ion chromatogram from the GC-MS gives information similar to that provided by a flame ionization detector (FID)-GC in that the peak area is proportional to the mass of the analytes detected. Total peak area and the peak areas of the individual analytes can be compared in the GC-MS case as long as the masses are in generally the same range. As discussed below, in some embodiments, purity of the Δ8-THC isolated by the process is greater than about 90%, 95%, 97% or 98%, or purity is greater that about 98% to 99%.

Chapter IV

In alternative embodiments, provided are continuous isolation and purification processes for preparing a substantially pure cannabinoids or a product enriched in Cannabis from plant or microbial material extracts. In alternative embodiments, provided herein are improved methods for purification and conversion processes based on a simple combination of continuous chromato-graphic gradient elutions. This exemplary process is simple, efficient and economic.

In alternative embodiments, provided are processes for producing and isolating cannabinoids from Cannabis and hemp extracts which contain cannabinoids in minute amounts. In alternative embodiments, provided are processes for producing and isolating cannabinoids from natural materials, including plant or plant extracts, microbes, or botanical drug substances, or synthetically and semi-synthetically prepared cannabinoid products, or from recombinantly engineered microbes, e.g., yeasts or bacteria recombinantly engineered to express one or more cannabinoids. In one embodiment, exemplary methods are inexpensive and provide specific cannabinoid concentrates (e.g., of CBD, Δ⁸-THC, Δ⁹-THC) of high purity.

In one embodiment, exemplary methods provide a simple and economical continuous process for separating and concentrating cannabinoids from solvent-extracted cannabinoid containing materials. In alternative embodiments, the solvent-extracted cannabinoid containing materials are derived from synthetic or biological materials such as hemp and Cannabis or botanical drug substances, or from microbial materials; and the solvent extraction methods can be polar solvent extractions, nonpolar solvent extractions, or the solvent extraction methods can comprise use of super critical carbon dioxide or mixtures thereof. The solvent extraction methods can extract cannabinoids substantially from the synthetic or biological, e.g., plant, matter, along with other plant matter comprising lipids, waxes, monoterpenes, sesquiterpenes, hydrocarbons, alkaloids, flavonoids and chlorophylls.

In alternative embodiments, methods provided herein comprise subjecting cannabinoid containing solvent extract starting materials to a number of chromatographic resins in various contacting steps using various gradient elution solutions.

In alternative embodiments, the cannabinoids from which can be fractionated and isolated using methods as provided herein, or which can be produced in reactions as provided herein, or which the solvent extracts are derived can be from, or can comprise: Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetrahydrocannabidiol (THCBD); tetrahydrocannabigerol (THCBG); tetrahydrocannabichromene (THCBC); tetrahydrocannabidivarol (THCBDV), or combinations thereof, including carboxylic acid precursors of the foregoing compounds and related naturally occurring compounds and their derivatives.

In alternative embodiments, provides are methods of preparing or obtaining a substantially pure cannabinoid or a product enriched in a given cannabinoid comprising:

-   -   (i) obtaining an extract or extract solution containing a         cannabinoid or a cannabinoid acid from a plant or microbial         material;     -   (ii) filtering the extract of step (i) to remove solids and         color bodies;     -   (iii) removing the extract solvent (as an extract fraction);     -   (v) continuously loading of an amount of extract solution over a         defined time increment over multiple stationary phase resins         columns such as a normal phase, reverse phase and/or ion         exchange chromatographic resin;     -   (vi) continuously eluting the extract solution using multiple         defined gradient elution solutions at specific time increments         and volumes;     -   (vii) continuously collecting the gradient elution fractions;     -   (viii) removal of the gradient elution solution from the         produced fractions to generate a substantially purified extract     -   (ix) optionally loading of specific first gradient elution         fractions on a reverse phase, ion exchange or normal         chromatographic resin;     -   (x) continuously eluting the first gradient elution fractions         with a second gradient elution solvent; and,     -   (xi) removing of the second gradient elution solvent from the         produced fractions to produce a purified extract.

In alternative embodiments, the methods further comprise:

-   -   a step (xii): loading of first and second gradient elution         solvents s onto an ion exchange chromatographic resin;     -   a step (xiii): eluting an extract solution from the ion exchange         chromatographic resin of step (xi) using a gradient solvent         solution and collecting the gradient elution fractions; and     -   (xiv) removing the gradient elution solution from the produced         fractions of step (xiii) to produce purified and substantially         purified extract.

In alternative embodiments, a “substantially pure” preparation of cannabinoid is defined as a preparation having a chromatographic purity (of the desired cannabinoid or cannabinoid acid) of greater than about 75%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99%, or greater than about 99.5%, or between about 70% and 99.9%, as determined by area normalization of an HPLC profile.

In alternative embodiments, the term “product enriched in a given cannabinoid” encompasses preparations having at least about 50%, or greater than about 75%, or greater than about 90%, 95% or 98%, or between about 50% and 99.9%, chromatographic purity, for the desired cannabinoid.

In alternative embodiments, a non-purified, or non-substantially purified, product can comprise a greater proportion of impurities, non-target materials and/or other cannabinoids than a “substantially pure” preparation. The cannabinoid can be (e.g., a cannabinoid purified or isolated by, or made by a reaction of, a method as provided herein can be): Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetra-hydrocannabidiol (THCBD); tetra-hydrocannabigerol (THCBG); tetra-hydrocannabichromene (THCBC); or, tetra-hydrocannabidivarol (THCBDV); the carboxylic acid precursors of the foregoing compounds; and related naturally occurring compounds and their derivatives.

In alternative embodiments, the term “cannabinoids” e.g., a cannabinoid purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,1′-di-methyl-pyrane ring, a variedly derivatized aromatic ring and/or a variedly unsaturated cyclohexyl ring and their immediate chemical precursors.

In alternative embodiments, the term “cannflavins”, e.g., a cannflavin purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,4-pyrone ring fused to a variedly derivatized aromatic ring and linked to a second variedly derivatized aromatic ring.

In alternative embodiments, the term “essential oils”, e.g., an essential oil used as a starting material in a process as provided herein, or an essential oil that may be isolated by a process as provided herein, includes or refers to a family of natural products that can contain a multiple of the 5-membered isoprene unit variedly substituted, often cyclized to form one or more ring systems; they may also contain series of aldehydes and/or ketones and esters of a variety of carboxylic acid substituted compounds.

In alternative embodiments, provided are methods for extracting and/or purifying cannabinoids from any plant or microbial material extract known to contain such cannabinoids, cannflavins and essential oils; and, optionally to purify cannflavins and to optionally purify essential oils. In alternative embodiments, the extract is passed through a series of chromatographic columns, for example, a normal phase column, a reversed phase column or an ion exchange column as a continuous simulated moving bed configuration.

In one embodiment, the chromatographic column is arranged for gradient elution fractioning using normal phase, reverse phase and/or ion exchange chromatography. In one embodiment Δ⁹-THC and CBD are fractionated out of the eluent. For example, in one embodiment, as the extract is passed over the column, Δ⁹-THC and CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THC and CBD. The fractions free of Δ⁹-THC and CBD are pooled, thereby producing an extract with Δ⁹-THC and CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, (substantially) only Δ⁹-THC is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extracts are pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be free of Δ⁹-THC. These fractions free of Δ⁹-THC are pooled, thereby producing an extract with Δ⁹-THC substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged for example 3, 4, 5, 6, 7, 8 or more, or between 3 and 30 or more, columns are arranged in a continuous rotation traveling through a series of contact points where gradient elution solutions and extract solution are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent is introduced, the second column moves to the fifth position where the fourth gradient is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced. The equipment is generally described in U.S. Pat. Nos. 4,764,276, 4,808,317, and 4,710,364, for example, each of which are expressly incorporated herein by reference.

In a particular embodiment the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent.

In some embodiments the fractions each produced fraction from the first continuous gradient elution separation is collected and the elution solution is removed. The collected fraction of cannabinoids is then loaded onto a normal phase, reverse phase or ion exchange resin and subject to the before mentioned continuous chromatography method to either preferentially purify a specific target cannabinoid from minor cannabinoids contained in the collected fraction.

In some embodiments, (substantially) only CBD is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of CBD. These fractions free of CBD are pooled, thereby producing an extract with CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, Δ⁹-THC carboxylic acid species or Δ⁹-THCA and CBD carboxylic acid species CBDA are fractionated out of out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a first elution are passed over the column, Δ⁹-THCA and CBDA are differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled first elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THCA and CBDA. These fractions (substantially) free of Δ⁹-THCA and CBDA are pooled, thereby producing an extract with Δ⁹-THCA and CBDA substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, the Δ⁹-THC may be eluted from the column, extracted or concentrated, for purifying, or substantially purifying, Δ⁹-THC. In alternative embodiments, the chromatographic column is arranged for continuous fractionation of a specific cannabinoid or groups of cannabinoids or their carboxylic acid species, cannflavin or essential oil or class of cannabinoids, cannflavins or essential oils out of the eluent, for example, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabidivarol (CBDV), tetrahydrocannabidiol (THCBD), tetrahydrocannabigerol (THCBG), tetrahydrocannabichromene (THCBC), tetrahydrocannabidivarol (THCBDV), Δ⁸-THC, the carboxylic acid precursors of the foregoing compounds, and related naturally occurring compounds and their derivatives. In alternate embodiments, the chromatographic column is arranged for continuous fractionation cannflavins and related naturally occurring compounds and their derivatives. In alternate embodiments, the system is arranged to continuously fractionate the components of essential oils. The list of compounds provided herein is not exhaustive and is in no way intended to be limiting. In these embodiments, the compound(s) of interest are retained or detained (e.g., reversibly bound) on the column so that fractions (alternatively, the last fractions) of the extract eluted from the column contain the compounds(s) of interest. In alternative embodiments, fractions containing the compound(s) of interest are pooled. In some embodiments, different compounds may be extracted with different solvents and then combined into a single extract. As will be appreciated by one knowledgeable in the art, in this manner, several different cannabinoids could be purified from a single extract.

In alternative embodiments, the “plant material or microbial” will be derived from one or more Cannabis or hemp plants, or from other plants, or from a microbial species, including fungi, lichen, yeast and bacteria. The term “plant material” encompasses a plant or plant part (e.g. bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries or parts thereof) as well as exudates, and includes material falling within the definition of “botanical raw material” in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research. The term “Cannabis plant(s)” encompasses wild type Cannabis sativa and also variants thereof, including Cannabis chemovars (varieties characterized by virtue of chemical composition) which naturally contain different amounts of the individual cannabinoids, also Cannabis sativa subspecies indica including the variants var. indica and var. kafiristanica, Cannabis indica and also plants which are the result of genetic crosses, self-crosses or hybrids thereof. The term “Cannabis plant material” also can encompass plant material derived from one or more Cannabis plants, and can comprise any “Cannabis plant material” including, e.g., herbal Cannabis and dried Cannabis biomass. The term “Cannabis plant material” also can encompass “decarboxylated Cannabis plant material”, which refers to Cannabis plant material which has been subject to a decarboxylation step in order to convert cannabinoid acids to the corresponding free cannabinoids.

In alternative embodiments, a starting material for a purification process as provided herein is an extract solution containing a cannabinoid or cannabinoid acid obtained from a natural or a synthetic source, e.g., a plant or microbial material. In alternative embodiments, the “extract solution containing a cannabinoid or cannabinoid acid” comprises a solvent extract of a plant or microbial material. Solvents used for extraction for use in the preparation of extract solutions can comprise non-polar solvents, polar solvents such as ethanol, methanol or water, or organic solvents such as liquid carbon dioxide, and combinations thereof. The solvent may be an organic solvent, selected from the group consisting of: non-polar solvents include liquid non-polar solvents comprising lower C1-C12, for example, C3 to C8, or straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol, methanol, water; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; or a supercritical fluid such as CO₂ with or without an organic solvent modifier.

In alternative embodiments, the extract is prepared by dissolving or partially dissolving the natural or synthetic, or the plant or microbial material in a solvent, removing insoluble material from the resultant solution (optionally by filtration with or without activated carbon, precipitation, centrifugation and the like), and optionally removing some or all of the extraction solvent from the solution (optionally by rotary evaporation) to form an extract or extract solution or concentrate containing a cannabinoid or cannabinoid acid.

In alternative embodiments, extractions can comprise using a technique referred to as accelerated solvent extraction or may use subcritical water or any combination of water and solvent. In one embodiment, when

isolating cannabinoid acids, a modified pH gradient elution solution is used. The primary purpose of this pH adjustment (the modified pH gradient) is to promote or prevent ionization of the cannabinoid acid. pH modified gradient elution solutions may be achieved by the additional of a small volume of acid or base to the solvent. It may be sufficient to add a relatively weak acid, such as acetic acid, oxalic acid, glycolic acid, carbonic acid or ammonium hydroxide or a small amount of base or buffering agent such as sodium hydroxide, magnesium hydroxide, sodium carbonate or sodium bicarbonate. For any given purification process the optimal amount and type of acid or base used may be determined empirically. An alternative exemplary acidified solvent is 0.1% acetic acid in ethanol or 0.1% sodium hydroxide in ethanol. In alternative embodiments, the neutralizing agent consists of for example sodium hydroxide, sodium carbonate, potassium carbonate, and potassium t-amylate, sodium bicarbonate.

Acidified non-polar and polar solvents of the types described above can be useful in preparation of gradient elutions using ion exchange chromatography. The solvents used in the conversion can comprise an organic solvent, e.g., a non-polar solvent, including a liquid non-polar solvent comprising lower C1-C12, or C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane, hexane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; a heterocyclic compound or cyclic ether for example, tetrahydrofuran and 2-Methyltetrahydrofuran and aromatic ring hydrocarbons such as benzene, toluene, xylene and ethylbenzene.

In alternative embodiments, the plant or microbial material is subjected to a decarboxylation step prior to solvent extraction. The purpose of the decarboxylation step is to convert cannabinoid acids present in the plant or microbial material to the corresponding free cannabinoids. In alternative embodiments, the decarboxylation is carried out by heating the plant or microbial material to a defined temperature for a suitable length of time. Decarboxylation of cannabinoid acids is a function of time and temperature, thus at higher temperatures a shorter period of time will be taken for complete decarboxylation of a given amount of cannabinoid acid. In alternative embodiments selecting appropriate conditions for decarboxylation consideration include minimizing thermal degradation of the desirable, pharmacological cannabinoids into undesirable degradation products, particularly thermal degradation of Δ⁹-THC. In alternative embodiments, the decarboxylation is carried out in a multi-step heating process in which the plant or microbial material is: i) heated to a first temperature for a first (relatively short) time period to evaporate off retained water and allow for uniform heating of the plant or microbial material; and ii) the temperature is increased to a second temperature for a second time period (typically longer than the first time period) until at least 95% conversion of the acid cannabinoids to their neutral form has occurred.

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” prepared from the starting plant or microbial material comprises a “botanical drug substance” prepared from the plant or microbial material, or a polar or non-polar solvent solution of such a botanical drug substance. In the context of this application a “botanical drug substance” is an extract derived from plant or microbial material, which extract fulfills the definition of “botanical drug substance” provided in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research of: “A drug substance derived from one or more plants, algae, or macroscopic fungi. It is prepared from botanical raw materials by one or more of the following processes: pulverization, decoction, expression, aqueous extraction, ethanolic extraction, or other similar processes.” “Botanical drug substances” derived from Cannabis plants include primary extracts prepared by such processes as, for example, maceration, percolation, and solvent extraction.

In alternative embodiments, solvent extraction may be carried out using essentially any solvent that dissolves, or substantially dissolves, cannabinoids/cannabinoid acids, such as for example C1 to C5 alcohols (e.g. ethanol, methanol), C5-C12 alkanes (e.g. hexane), norflurane, or 1,1,1,2-tetrafluoroethane (HFA134a), 1,1,1,2,3,3,3-Heptafluoropropane (or HFA227), chloroform, dichloromethane, dichloroethane and carbon dioxide. When solvents such as those listed above are used, the resultant extract typically contains non-specific lipid-soluble material. This can optionally be removed by a variety of processes including filtration to remove solids, “winterization”, which involves for example chilling to −20° C. or lower followed by filtration to remove waxy ballast, extraction with liquid carbon dioxide and by distillation.

In alternative embodiments, any protocol for the preparation of botanical drug substances from Cannabis and hemp plant material can be used, e.g., as described in International patent application WO 02/064109. In alternative embodiments, the botanical drug substance is obtained by carbon dioxide (CO₂) extraction, polar solvent extraction or non-polar solvent extraction or combinations thereof followed by a filtration. Optionally a secondary extraction is performed to remove a substantial proportion of non-cannabinoid materials, e.g. waxes, wax esters and glycerides, unsaturated fatty acid residues, terpenes, carotenes, and flavonoids and other ballast.

In alternative embodiments, if it is intended to prepare free cannabinoids from the plant or microbial material (e.g., Cannabis), then the material is heated to a defined temperature for a defined period of time in order to partially or substantially decarboxylate cannabinoid acids to free cannabinoids prior to extraction of the botanical drug substance. In alternative embodiments, the botanical drug substance is prepared according to a process comprising the following steps: i) optional decarboxylation of the plant material, ii) extraction with polar or non-polar solvent, to produce a crude botanical drug substance, iii) optional precipitation with C1-C5 alcohol to reduce the proportion of non-target materials, iv) removal of the precipitate (for example, by filtration, precipitation, centrifugation and the like), v) optional treatment with activated charcoal, and vi) evaporation to remove C1-C5 alcohol and water, thereby producing a final botanical drug substance.

In alternative embodiments, the tetrahydrocannabinol at therapeutically effective concentrations or dosages is combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, methacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients that can be used include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethyl cellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethycellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example, Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed.

In alternative embodiments, purity is determined by Gas Chromatography-Mass Spectrometry (GC-MS) and/or by analytical high-performance liquid chromatography (HPLC). The total ion chromatogram from the GC-MS gives information similar to that provided by a flame ionization detector (FID)-GC in that the peak area is proportional to the mass of the analytes detected. Total peak area and the peak areas of the individual analytes can be compared in the GC-MS case as long as the masses are in generally the same range. As discussed below, in some embodiments, purity of the isolated cannabinoids by the continuous process is greater than about 90%, 95%, 97% or 98%, or purity is greater that about 98% to 99%.

Chapter V

In alternative embodiments, provided are continuous isolation and purification processes for preparing a substantially pure cannabidiol or a product enriched in cannabidiol from plant material extracts. In alternative embodiments, provided herein are improved methods for converting cannabidiol (CBD) to Δ⁸-THC and Δ⁹-THC, including a purification and conversion process based on a simple combination of continuous chromato-graphic gradient elutions and semi continuous isomerization reactions. This exemplary process is simple, efficient and economic.

In alternative embodiments, provided are methods of preparing cannabinoids in substantially pure form starting from plant extract material and conversion of the purified CBD to form both Δ⁸ THC and Δ⁹ THC and subsequent purification of the produced Δ⁸ tetrahydrocannabinol (Δ⁸ THC) into Δ⁹ tetrahydrocannabinol (Δ⁹ THC) using continuous chromatography.

In alternative embodiments, provided are processes for producing and isolating cannabinoids from Cannabis and hemp extracts which contain cannabinoids in minute amounts. In alternative embodiments, provided are processes for producing and isolating cannabinoids from natural materials, including plant or plant extracts, microbes, or botanical drug substances, or synthetically and semi-synthetically prepared cannabinoid products, or from recombinantly engineered microbes, e.g., yeasts or bacteria recombinantly engineered to express one or more cannabinoids. In one embodiment, exemplary methods are inexpensive and provide specific cannabinoid concentrates (e.g., of CBD, Δ⁸-THC, Δ⁹-THC) of high purity.

In one embodiment, exemplary methods provide a simple and economical continuous process for separating and concentrating cannabinoids from solvent-extracted cannabinoid containing materials. In one embodiment, exemplary methods provide a method that first converts the substantially isolated CBD into a mixture of Δ⁸-THC and Δ⁹-THC, and then subsequently purifying or isolating the Δ⁸-THC and Δ⁹-THC. In alternative embodiments, the solvent-extracted cannabinoid containing materials are derived from synthetic or biological materials such as hemp and Cannabis or botanical drug substances, or from microbial materials; and the solvent extraction methods can be polar solvent extractions, nonpolar solvent extractions, or the solvent extraction methods can comprise use of super critical carbon dioxide or mixtures thereof. The solvent extraction methods can extract cannabinoids substantially from the synthetic or biological, e.g., plant, matter, along with other plant matter comprising lipids, waxes, monoterpenes, sesquiterpenes, hydrocarbons, alkaloids, flavonoids and chlorophylls.

In alternative embodiments, methods provided herein comprise subjecting cannabinoid containing solvent extract starting materials to a number of chromatographic resins in various contacting steps using various gradient elution solutions.

In alternative embodiments, the cannabinoids which can be fractionated and isolated using methods as provided herein, or which can be produced in reactions as provided herein, or from which the solvent extracts are derived can be from, or can comprise: Δ⁸tetrahydrocannabinol (Δ⁸ THC); Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetrahydrocannabidiol (THCBD); tetrahydrocannabigerol (THCBG); tetrahydrocannabichromene (THCBC); tetrahydrocannabidivarol (THCBDV), or combinations thereof, including carboxylic acid precursors of the foregoing compounds and related naturally occurring compounds and their derivatives.

In alternative embodiments, provides are methods of preparing or obtaining a substantially pure cannabinoid or a product enriched in a given cannabinoid comprising:

-   -   (i) obtaining or providing, or having provided, an extract or         extract solution, or an aliquot or sample, containing a         cannabinoid or a cannabinoid acid from a natural or the         synthetic source, e.g., a plant material;     -   (ii) optionally filtering the extract or extract solution, or         aliquot or sample, of step (i) to remove solids and color         bodies;     -   (iii) optionally removing the extract solvent (as an extract         fraction);     -   (iv) continuously loading of an amount of extract solution over         a defined time increment over multiple stationary phase resins         columns such as a normal phase, reverse phase and/or ion         exchange chromatographic resin;     -   (v) continuously eluting the extract, or extract solution, or         aliquot or sample, solution using multiple defined gradient         elution solutions at specific time increments and volumes;     -   (vi) continuously collecting the gradient elution fractions;     -   (vii) removal of the gradient elution solution from the produced         fractions to generate a substantially purified extract, or a         substantially purified extract from each fraction;     -   (viii) optionally loading of specific first gradient elution         fractions on a reverse phase, ion exchange or normal         chromatographic resin;     -   (ix) continuously eluting the first gradient elution fractions         with a second gradient elution solvent; and,     -   (x) removing of the second gradient elution solvent from the         produced fractions to produce a purified extract, or a         substantially purified extract from each fraction.

In alternative embodiments, the methods further comprise:

-   -   a step (xi): loading of first and second gradient elution         solvents s onto an ion exchange chromatographic resin;     -   a step (xii): eluting an extract solution from the ion exchange         chromatographic resin of step (x) using a gradient solvent         solution and collecting the gradient elution fractions; and     -   (xiii) removing the gradient elution solution from the produced         fractions of step (xiv) to produce purified and substantially         purified extract.

In alternative embodiments, provided are methods of substantially converting CBD to Δ⁸ THC and Δ⁹ THC comprising:

-   -   (a) providing a reaction mixture comprising a catalyst in an         organic solvent;     -   (b) adding CBD material from steps (viii) and steps (xiii),         above;     -   (c) mixing said reaction mixture;     -   (d) reacting mixture for a period of time at a controlled         temperature;     -   (e) adding a base to the reaction mixture;     -   (f) allowing the mixture to separate into an aqueous phase and         an organic phase;     -   (g) removing the organic phase; and,     -   (h) loading the organic phase onto a normal phase chromatography         column     -   (i) eluting the organic phase with an organic solvent and         recovering substantially pure CBD, Δ⁸ THC and Δ⁹ THC     -   optionally, repeating steps (a) through (i).

In alternative embodiments, a “substantially pure” preparation of cannabinoid is defined as a preparation having a chromatographic purity (of the desired cannabinoid or cannabinoid acid) of greater than about 75%, or greater than about 96%, or greater than about 97%, or greater than about 98%, or greater than about 99%, or greater than about 99.5%, or between about 70% and 99.9%, as determined by area normalisation of an HPLC profile.

In alternative embodiments, the term “product enriched in a given cannabinoid” encompasses preparations having at least about 50%, or greater than about 75%, or greater than about 90%, 95% or 98%, or between about 50% and 99.9%, chromatographic purity, for the desired cannabinoid.

In alternative embodiments, the term “about” is within 20% of the stated value, or 19%, or 18%, or 17%, or 16%, or 15%, or 14%, or 13%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, or 0.5%, or 0.1%, or 0.05%, or 0.01%, or is between 20% and 0.01% of the stated value.

In alternative embodiments, a non-purified, or non-substantially purified, product can comprise a greater proportion of impurities, non-target materials and/or other cannabinoids than a “substantially pure” preparation. The cannabinoid can be (e.g., a cannabinoid purified or isolated by, or made by a reaction of, a method as provided herein can be): Δ⁸tetrahydrocannabinol (Δ⁸ THC); Δ⁹tetrahydrocannabinol (Δ⁹ THC); cannabidiol (CBD); cannabinol (CBN); cannabigerol (CBG); cannabichromene (CBC); cannabidivarol (CBDV); tetra-hydrocannabidiol (THCBD); tetra-hydrocannabigerol (THCBG); tetra-hydrocannabichromene (THCBC); or, tetra-hydrocannabidivarol (THCBDV); the carboxylic acid precursors of the foregoing compounds; and related naturally occurring compounds and their derivatives.

In alternative embodiments, the term “cannabinoids” e.g., a cannabinoid purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,1′-di-methyl-pyrane ring, a variedly derivatized aromatic ring and/or a variedly unsaturated cyclohexyl ring and their immediate chemical precursors.

In alternative embodiments, the term “cannflavins”, e.g., a cannflavin purified or isolated by, or made by a reaction of a process as provided herein, includes or refers to a family of natural products that can contain a 1,4-pyrone ring fused to a variedly derivatized aromatic ring and linked to a second variedly derivatized aromatic ring.

In alternative embodiments, the term “Lewis acid” refers to a powerful electron pair acceptor; and examples include but are by no means limited to BF₃Et₂O (boron trifluoride ditheyl etherate), p-toluenesulfonic acid and boron trifluoride.

In alternative embodiments, the term “non-oxidizing acid” refers to hydrobromic, hydrochloric, hydrofluoric, acetic, benzoic, chloroacetic, formic, phosphoric, sulfuric, trifluroacetic and oxalic acids.

In alternative embodiments, the term “essential oils”, e.g., an essential oil used as a starting material in a process as provided herein, or an essential oil that may be isolated by a process as provided herein, includes or refers to a family of natural products that can contain a multiple of the 5-membered isoprene unit variedly substituted, often cyclized to form one or more ring systems; they may also contain series of aldehydes and/or ketones and esters of a variety of carboxylic acid substituted compounds.

In alternative embodiments, provided are methods for extracting and/or purifying cannabinoids from any plant material extract known to contain such cannabinoids, cannflavins and essential oils; and, optionally to purify cannflavins and to optionally purify essential oils. In alternative embodiments, the extract is passed through a series of chromatographic columns, for example, a normal phase column, a reversed phase column or an ion exchange column as a continuous simulated moving bed configuration.

In one embodiment, the chromatographic column is arranged for gradient elution fractioning using normal phase, reverse phase and/or ion exchange chromatography. In one embodiment Δ⁹-THC and CBD are fractionated out of the eluent. For example, in one embodiment, as the extract is passed over the column, Δ⁹-THC and CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract comes off the column after gradient elution transition, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THC and CBD. The fractions free of Δ⁹-THC and CBD are pooled, thereby producing an extract with Δ⁹-THC and CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, (substantially) only Δ⁹-THC is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially retained or detained (e.g., reversibly bound) on the column. As the extracts pooled from previous elution fractions come off the column, the initial fractions eluted off the column will be free of Δ⁹-THC. These fractions free of Δ⁹-THC are pooled, thereby producing an extract with Δ⁹-THC substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged, for example, 3, 4, 5, 6, 7, 8, 9, or 10 or more, or between 3 and 30, columns are arranged in a continuous rotation traveling through a series of contact points where gradient elution solutions and extract solution are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent n is introduced, the second column moves to the fifth position where the fourth gradient solvent is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced. In a particular embodiment the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent.

In some embodiments, when using a continuous chromatography apparatus or device, a series of columns are arranged, for example, 3, 4, 5, 6, 7, 8, 9, or 10 or more, or between 3 and 30, columns are arranged in a continuous rotation traveling through a series of contact points where gradient elution solutions and extract solution are introduced at fixed points for a period of time allowing for continuous loading and elution, and collection of fractions. The first column is loaded with extract solution at the first position (or station). The first column is then moved to the second position where the first gradient elution is introduced (or loaded) while at the same time the second column is loaded with extract solution at position one. The first column then rotates (i.e., is moved) to the third position where the second gradient solvent is introduced, the second column moves to the second position where the first gradient solvent is introduced and the third column is loaded with extract solution at position one. The first column then moves to the fourth position where the third gradient solvent is introduced, the second column moves to the third position where the second gradient solvent is introduced, the third column moves to the second position where the first gradient solvent is introduced and the fourth column is loaded with extract solution at position one. The first column then moves to the fifth position where the fourth gradient solvent is introduced, the second column moves to the fourth position where the third gradient solvent is introduced, the third column moves to the third position where the second gradient solvent is introduced, the fourth column moves to the second position where the first gradient solvent is introduced and the fifth column is loaded with extract solution at position one. The first column then moves to the sixth position where the fifth gradient solvent is introduced, the second column moves to the fifth position where the fourth gradient solvent is introduced, the third column moves to the fourth position where the third gradient solvent is introduced, the fourth column moves to the third position where the second gradient solvent is introduced, the fifth column moves to the second position where the first gradient solvent is introduced and the sixth column is loaded with extract solution at position one. The first column then moves or returns to the first position where extract solution is loaded, the second column moves to the sixth position where the fifth gradient solvent is introduced, the third column moves to the fifth position where the fourth gradient solvent is introduced, the fourth column moves to the fourth position where the third gradient solvent is introduced, the fifth column moves to the third position where the second gradient solvent is introduced and the sixth column moves to the second position where the first gradient solvent is introduced.

In a particular embodiment the first gradient solvent elutes CBD, CBG and CBN and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD, CBG and CBN is differentially produced in the eluent (e.g., by use of a gradient elution process). In a particular embodiment second gradient solvent elutes CBD and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially produced in the eluent. In a particular embodiment the third gradient solvent elutes CBD and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD and Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fourth gradient elutes Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, Δ⁹-THC is differentially produced in the eluent. In a particular embodiment the fifth gradient solvent elutes CBC, THC-A, terpenes and Δ⁹-THC and is substantially fractionated in the eluent. For example, in one embodiment, as the extract or pooled fractions from a normal phase, reverse phase and/or ion exchange elution are passed over the column, CBC, THC-A, terpenes and Δ⁹-THC is differentially produced in the eluent. The second gradient solvent containing CBD is combined with a Lewis acid or non-oxidizing acid catalyst, optionally additional CBD or substantially pure CBD and/or (Δ8-THC) and/or Δ9-tetrahydrocannabinol (Δ9-THC) can be added to the reaction mixture, mixing said reaction mixture for a period of time; adding a neutralizing agent to said mixture; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally adding organic phase to a chromatography column and eluting the (Δ8-THC) and/or Δ9-tetrahydrocannabinol (Δ9-THC) from the organic phase. The tetrahydrocannabinol may then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase.

In some embodiments, (substantially) only CBD is substantially fractionated out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a previous normal phase, reverse phase and/or ion exchange elution are passed over the column, CBD is differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled previous elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of CBD. These fractions free of CBD are pooled, thereby producing an extract with CBD substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, Δ⁹-THC carboxylic acid species or Δ⁹-THCA and CBD carboxylic acid species CBDA are fractionated out of out of the eluent. For example, in one embodiment, as the extract or pooled fractions from a first elution are passed over the column, Δ⁹-THCA and CBDA are differentially retained or detained (e.g., reversibly bound) on the column. As a result, as the extract or pooled first elution fractions comes off the column, the initial fractions eluted off the column will be (substantially) free of Δ⁹-THCA and CBDA. These fractions (substantially) free of Δ⁹-THCA and CBDA are pooled, thereby producing an extract with Δ⁹-THCA and CBDA substantially removed (e.g., in alternative embodiments, “substantially removed” or “substantially fractionated” means at least 85%, 90%, 95%, 98%, 99% or 99.5% or more removed or fractionated).

In some embodiments, the Δ⁹-THC may be eluted from the column, extracted or concentrated, for purifying, or substantially purifying, Δ⁹-THC. In alternative embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) a specific cannabinoid or groups of cannabinoids or their carboxylic acid species, cannflavin or essential oil or class of cannabinoids, cannflavins or essential oils out of the eluent, for example, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabidivarol (CBDV), tetrahydrocannabidiol (THCBD), tetrahydrocannabigerol (THCBG), tetrahydrocannabichromene (THCBC), tetrahydrocannabidivarol (THCBDV), Δ⁸-THC, the carboxylic acid precursors of the foregoing compounds, and related naturally occurring compounds and their derivatives. In alternate embodiments, the chromatographic column is arranged for fractionating (e.g., sequentially fractionating) cannflavins and related naturally occurring compounds and their derivatives. In alternate embodiments, the system is arranged to fractionate the components of essential oils. The list of compounds provided herein is not exhaustive and is in no way intended to be limiting. In these embodiments, the compound(s) of interest are retained or detained (e.g., reversibly bound) on the column so that fractions (alternatively, the last fractions) of the extract eluted from the column contain the compounds(s) of interest. In alternative embodiments, fractions containing the compound(s) of interest are pooled. In some embodiments, different compounds may be extracted with different solvents and then combined into a single extract. As will be appreciated by one knowledgeable in the art, in this manner, several different cannabinoids could be purified from a single extract.

In alternative embodiments, the “plant material” or botanical drug substance is derived from one or more Cannabis or hemp plants, or from other plants, or a microbial source, including yeast, bacteria, lichen, algae. The term “plant material” encompasses a plant or plant part (e.g. bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries or parts thereof) as well as exudates, and includes material falling within the definition of “botanical raw material” in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research. The term “Cannabis plant(s)” encompasses wild type Cannabis sativa and also variants thereof, including Cannabis chemovars (varieties characterized by virtue of chemical composition) which naturally contain different amounts of the individual cannabinoids, also Cannabis sativa subspecies indica including the variants var. indica and var. kafiristanica, Cannabis indica and also plants which are the result of genetic crosses, self-crosses or hybrids thereof. The term “Cannabis plant material” also can encompass plant material derived from one or more Cannabis plants, and can comprise any “Cannabis plant material” including, e.g., herbal Cannabis and dried Cannabis biomass. The term “Cannabis plant material” also can encompass “decarboxylated Cannabis plant material”, which refers to Cannabis plant material which has been subject to a decarboxylation step in order to convert cannabinoid acids to the corresponding free cannabinoids.

In alternative embodiments, a starting material for a purification process as provided herein is an extract solution containing a cannabinoid or cannabinoid acid obtained from a natural or a synthetic source, e.g., a plant or a microbial material. In alternative embodiments, the “extract solution containing a cannabinoid or cannabinoid acid” comprises a solvent extract of a plant material. Solvents used for extraction for use in the preparation of extract solutions can comprise non-polar solvents, polar solvents such as ethanol, methanol or water, or organic solvents such as liquid carbon dioxide, and combinations thereof. The solvent may be an organic solvent, selected from the group consisting of: non-polar solvents include liquid non-polar solvents comprising lower C1-C12, for example, C3 to C8, or straight chain or branched chain alkanes, for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane; a low molecular weight alcohol, polar solvents comprising, for example, ethanol, methanol, water; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane or mixtures thereof; or a supercritical fluid such as CO₂ with or without an organic solvent modifier.

In alternative embodiments, the extract is prepared by dissolving or partially dissolving natural or synthetic, or the plant or microbial material, in a solvent, removing insoluble material from the resultant solution (optionally by filtration with or without activated carbon, precipitation, centrifugation and the like), and optionally removing some or all of the extraction solvent from the solution (optionally by evaporation, e.g., rotary evaporation) to form an extract or extract solution or concentrate containing a cannabinoid or cannabinoid acid.

In alternative embodiments, extractions can comprise using a technique referred to as accelerated solvent extraction or may use subcritical water or any combination of water and solvent. In one embodiment, when

isolating cannabinoid acids, a modified pH gradient elution solution is used. The primary purpose of this pH adjustment (the modified pH gradient) is to promote or prevent ionization of the cannabinoid acid. pH modified gradient elution solutions may be achieved by the additional of a small volume of acid or base to the solvent. It may be sufficient to add a relatively weak acid, such as acetic acid, oxalic acid, glycolic acid, carbonic acid or ammonium hydroxide or a small amount of base or buffering agent such as sodium hydroxide, magnesium hydroxide, sodium carbonate or sodium bicarbonate. For any given purification process the optimal amount and type of acid or base used may be determined empirically. An alternative exemplary acidified solvent is 0.1% acetic acid in ethanol or 0.1% sodium hydroxide in ethanol. In alternative embodiments, the neutralizing agent consists of for example sodium hydroxide, sodium carbonate, potassium carbonate, and potassium t-amylate, sodium bicarbonate.

Acidified non-polar and polar solvents of the types described above can be useful in preparation of gradient elutions using ion exchange chromatography. The solvents used in the conversion can comprise an organic solvent, e.g., a non-polar solvent, including a liquid non-polar solvent comprising lower C1-C12, or C3 to C8, straight chain or branched chain alkanes for example, methane, ethane, propane, butane, pentane, toluene, trimethylpentane, hexane; a low molecular weight alcohol, polar solvents consisting of for example, ethanol; a low molecular weight chlorinated hydrocarbon, for example, chloroform and dichloromethane; a heterocyclic compound or cyclic ether for example, tetrahydrofuran and 2-Methyltetrahydrofuran and aromatic ring hydrocarbons such as benzene, toluene, xylene and ethylbenzene.

In alternative embodiments, the plant material is subjected to a decarboxylation step prior to solvent extraction. The purpose of the decarboxylation step is to convert cannabinoid acids present in the plant material to the corresponding free cannabinoids. In alternative embodiments, the decarboxylation is carried out by heating the plant material to a defined temperature for a suitable length of time. Decarboxylation of cannabinoid acids is a function of time and temperature, thus at higher temperatures a shorter period of time will be taken for complete decarboxylation of a given amount of cannabinoid acid. In alternative embodiments selecting appropriate conditions for decarboxylation consideration include minimizing thermal degradation of the desirable, pharmacological cannabinoids into undesirable degradation products, particularly thermal degradation of Δ⁹-THC. In alternative embodiments, the decarboxylation is carried out in a multi-step heating process in which the plant material is: i) heated to a first temperature for a first (relatively short) time period to evaporate off retained water and allow for uniform heating of the plant material; and ii) the temperature is increased to a second temperature for a second time period (typically longer than the first time period) until at least 95% conversion of the acid cannabinoids to their neutral form has occurred.

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” prepared from the starting plant material comprises a “botanical drug substance” prepared from the plant material, or a polar or non-polar solvent solution of such a botanical drug substance. In the context of this application a “botanical drug substance” is an extract derived from plant material, which extract fulfills the definition of “botanical drug substance” provided in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research of: “A drug substance derived from one or more plants, algae, or macroscopic fungi. It is prepared from botanical raw materials by one or more of the following processes: pulverization, decoction, expression, aqueous extraction, ethanolic extraction, or other similar processes.” “Botanical drug substances” derived from Cannabis plants include primary extracts prepared by such processes as, for example, maceration, percolation, and solvent extraction.

In alternative embodiments, solvent extraction may be carried out using essentially any solvent that dissolves, or substantially dissolves, cannabinoids/cannabinoid acids, such as for example C1 to C5 alcohols (e.g. ethanol, methanol), C5-C12 alkanes (e.g. hexane), norflurane (HFA134a), 1,1,1,2,3,3,3-Heptafluoropropane (or HFA227), chloroform, dichloromethane, dichloroethane and carbon dioxide. When solvents such as those listed above are used, the resultant extract typically contains non-specific lipid-soluble material. This can optionally be removed by a variety of processes including filtration to remove solids, “winterization”, which involves for example chilling to −20° C. or lower followed by filtration to remove waxy ballast, extraction with liquid carbon dioxide and by distillation.

In alternative embodiments, any protocol for the preparation of botanical drug substances from Cannabis and hemp plant material can be used, e.g., as described in International patent application WO 02/064109. In alternative embodiments, the botanical drug substance is obtained by carbon dioxide (CO₂) extraction, polar solvent extraction or non-polar solvent extraction or combinations thereof followed by a filtration. Optionally a secondary extraction is performed to remove a substantial proportion of non-cannabinoid materials, e.g. waxes, wax esters and glycerides, unsaturated fatty acid residues, terpenes, carotenes, and flavonoids and other ballast.

In alternative embodiments, if it is intended to prepare free cannabinoids from the plant or microbial material, e.g., Cannabis, then the material is heated to a defined temperature for a defined period of time in order to partially or substantially decarboxylate cannabinoid acids to free cannabinoids prior to extraction of the botanical drug substance. In alternative embodiments, the botanical drug substance is prepared according to a process comprising the following steps: i) optional decarboxylation of the plant material, ii) extraction with polar or non-polar solvent, to produce a crude botanical drug substance, iii) optional precipitation with C1-C5 alcohol to reduce the proportion of non-target materials, iv) removal of the precipitate (for example, by filtration, precipitation, centrifugation and the like), v) optional treatment with activated charcoal, and vi) evaporation to remove C1-C5 alcohol and water, thereby producing a final botanical drug substance.

In alternative embodiments, provided are methods for converting substantially purified cannabidiol (CBD) to Δ8-tetrahydrocannabinol (Δ8-THC) and Δ9-tetrahydrocannabinol (Δ9-THC). As will be appreciated by one knowledgeable in the art and as discussed below, the reaction times may be varied somewhat, producing product at different yields and purities. Furthermore, functional equivalents may be substituted where appropriate.

In alternative embodiments, an exemplary method of converting CBD to Δ8-tetrahydrocannabinol (Δ8-THC) and Δ9-tetrahydrocannabinol (Δ9-THC) comprises: providing a reaction mixture comprising a Lewis acid or non-oxidizing acid catalyst in a reaction solvent, adding a substantially pure CBD or substantially pure CBD and/or (Δ8-THC) and/or Δ9-tetrahydrocannabinol (Δ9-THC) to the reaction mixture, mixing said reaction mixture for a period of time; adding a neutralizing agent to said mixture; filtration of catalyst and neutralizing agent from mixture; optionally allowing mixture to separate into an aqueous and organic phase; optionally removing the reaction solvent; optionally dissolving organic phase in a second solvent; adding organic phase to a chromatography column and eluting the tetrahydrocannabinol from the organic phase. The tetrahydrocannabinol may then be combined with suitable excipients known in the art, thereby forming a pharmaceutical composition. In one embodiment, the mixture is allowed to separate into an aqueous phase and an organic phase; and optionally the process further comprises removing the organic phase).

In alternative embodiments, the tetrahydrocannabinol at therapeutically effective concentrations or dosages is combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, methacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients that can be used include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethyl cellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethycellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example, Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed.

In some embodiments, the catalyst is a Lewis acid, for example, p-toluenesulfonic acid, boron trifluoride or BF₃Et₂O. In some embodiments, the BF₃Et₂O (boron trifluoride diethyl etherate) is in dry methylene chloride, ethyl acetate, ethanol, hexane or other organic solvent. In yet other examples, the catalyst may be hydrochloric acid in ethanol or sulfuric acid in cyclohexane.

In some embodiments, the catalyst is a non-oxidizing acid, for example, formic acid, acetic acid or hydrobromic acid. In some embodiments, the non-oxidizing acid is in dry methylene chloride, ethyl acetate, ethanol, hexane or other organic solvent.

In some embodiments, a base is added to the reaction mixture prior to optionally allowing the reaction mixture to separate into organic and aqueous phases. The base may be an alkali metal hydrogen carbonate, carbonate of an alkali metal, lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)), calcium hydroxide (Ca(OH)), strontium hydroxide (Sr(OH)), barium hydroxide (Ba(OH)).

In some embodiments, the organic layer is dried prior to eluting. In these embodiments, a suitable drying or dehydration compound, for example, MgSO4 or Na₂SO₄ is used.

In yet other embodiments, the process may be carried out under an inert atmosphere such as a nitrogen (e.g., N₂) atmosphere.

In alternative embodiments, and as discussed below, yield is determined by looking at the peak area for the isolated compound in the gas chromatography-mass spectra analysis of the crude reaction product mixture and the final reaction product mixture. It is important to note that in the prior art, yield is often calculated on the basis of first isolated crude product before final purification. In some embodiments of processes provided herein yield of Δ8-THC and Δ9-THC is at least about 75%; in other embodiments, the yield of Δ8-THC and Δ9-THC is at least about 90%; and in other embodiments, yield of Δ8-THC and Δ9-THC is at least about 98%; and in yet other embodiments, yield of Δ8-THC and Δ9-THC is between about 75 to 98% or 99%.

In alternative embodiments, purity is determined by Gas Chromatography-Mass Spectrometry (GC-MS) and/or by analytical high-performance liquid chromatography (HPLC). The total ion chromatogram from the GC-MS gives information similar to that provided by a flame ionization detector (FID)-GC in that the peak area is proportional to the mass of the analytes detected. Total peak area and the peak areas of the individual analytes can be compared in the GC-MS case as long as the masses are in generally the same range. As discussed below, in some embodiments, purity of the Δ8-THC and Δ9-THC mixture isolated by the process is greater than about 90%, 95%, 97% or 98%, or purity is greater that about 98% to 99%.

Continuous Chromatography Apparatus or Devices

In alternative embodiments, processes and methods as provided herein comprise use of continuous chromatography apparatus or devices, and associated robots and software. Any continuous chromatography apparatus or device known in the art can be used, e.g., as described in: Challener, BioPharm International, Vol 31, Issue 4, pg 14-18; Zobel et al Ind. Eng. Chem. Res., 2014, 53 (22), pp 9169-9185; Horvath et al Org. Process Res. Dev., 2015, 19 (6), pp 624-634; or as manufactured

-   -   e.g., by Ionex, Calgon, ThermoFischer Scientific.

Embodiments as provided herein will be further described with reference to the Examples described herein; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES Example 1: Exemplary Methods

In alternative embodiments, the “extract, aliquot or sample comprising or containing a cannabinoid or a cannabinoid acid” is dissolved in a gradient elution solution and subjected to a chromatographic purification step to produce substantially pure cannabinoids and a product enriched in a given cannabinoid. The purpose of this step is to first remove the targeted non-cannabinoid material comprising for example terpenes, carotenes, and flavonoids and also to provide a degree of separation/fractionation of the various cannabinoid/cannabinoid acid components of the extract.

In alternative embodiments, the product of the gradient elution chromatographic step is collected in multiple fractions, which may then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid and/or cannabinoid acid may then be selected for further purification. Optionally the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent solution is added to change the polarity or hydrogen potential of the gradient solvent solution contained in the selected fraction. The selected fraction can then be subjected to one or more additional chromatographic steps and one or more gradient elution solutions modifications.

In alternative embodiments, the chromatographic step comprises a column chromatography in a fixed or a continuous mode, and is can be based on molecular sizing, polarity and/or hydrogen potentiality. In alternative embodiments, the column matrix materials are, for example; silica (or silica gel) and/or alumina normal phase and reverse phase such as for example C18, C8, C4, C2, amino, cyano, phenyl, diol, WAX, SAX, WCX, SCX, Thiol; acidic, basic and neutral, styrenic, brominated styrenic; ion exchange resins such as strongly acidic, typically featuring sulfonic acid groups, e.g. sodium polystyrene sulfonate or polyAMPS; strongly basic, typically featuring quaternary amino groups, for example, trimethylammonium groups, e.g. polyAPTAC); weakly acidic, typically featuring carboxylic acid groups; weakly basic, typically featuring primary, secondary, and/or tertiary amino groups, e.g. polyethylene amine.

In alternative embodiments, various different elution solutions are used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, hexane, butane, pentane, heptane, octane, carbon tetrachloride, etc.

In alternative embodiments, the chromatographic step comprises column chromatography on for example, a silica (or silica gel) or an alumina column, and the step can comprise eluting with a 5:1; 4:1; 3:1; 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1:1; 0.9:1; 0.5:1; 0.1:1 mixture of elution solutions such as chloroform, methylene dichloride, ethylene dichloride, methanol, ethanol, propanol and/or water. Any suitable combination of normal phase column packing material and solvent having separation characteristics suitable for use in separation (fractionation) of cannabinoids and/or cannabinoid acids can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. The fractions are tested for the presence of the desired cannabinoid and/or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid and/or cannabinoid acid selected for further processing. Solvent can be then removed from the selected fractions, for example, by evaporation, e.g., rotary evaporation or equivalents.

In alternative embodiments, the fractions enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed.

In alternative embodiments, the fractions containing a product enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid such as mixtures comprising CBD and THC, or mixtures of CBD and CBG or mixtures of CBG and CBC, are dissolved in a gradient elution solution and subjected to a reverse phase chromatographic purification step to produce substantially pure extracts.

In alternative embodiments, the product of the reverse phase chromatographic step is collected in multiple fractions, which may then be tested for the presence of the desired cannabinoid and/or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid may then be selected for further purification. In alternative embodiments, the elution solution is removed from the selected fractions or optionally additional solution is added to change the polarity of the solution contained in the selected fraction.

An exemplary embodiment comprises: fractions enriched in a given cannabinoid obtained from the elution of a reverse phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed. In alternative embodiments, the selected fraction is then subjected to one or more additional reverse phase chromatographic steps and one or more elution solutions modifications. In alternative embodiments, the reverse phase chromatographic step comprises a column chromatography in fixed or continuous mode, and can be based on polarity. Examples of reverse phase column chromatography include but are not limited to pure silica, alkyl chain-bonded silica, cyano-bonded silica, and phenyl-bonded silica.

In alternative embodiments, various different elution solutions may be used in combination with this type of matrix, for example dimethyl sulfoxide, water, dimethylformamide, methanol, saline chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, dichloroethane, etc.

In alternative embodiments, the chromatographic step comprises column chromatography using an ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™ column, and eluting can be with a 20:1; 15:1; 10:1, 5:1; 4.5:1; 4:1; 3.5:1; 3:1 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1.001:1; 0.1:1 mixture of elution solvents such as chloroform, dichloromethane, dichloroethane, ethanol, propanol, dimethyl sulfoxide, water, dimethylformamide, methanol, saline chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, dichloroethane, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, and the like, and/or water. Any suitable combination of ion exchange phase column packing material and solvent having separation characteristics suitable for use in purification of cannabinoids and cannabinoids acid can be used with equivalent effect.

In alternative embodiments, the column eluate is collected in several fractions. In alternative embodiments, the fractions are tested for the presence of the desired cannabinoid or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid or cannabinoid acid. In alternative embodiments, solvent is then removed from the selected fractions, optionally by evaporation, e.g., rotary evaporation.

In alternative embodiments, the fractions containing a product are enriched in a given cannabinoid acid obtained from the elution chromatographic step or steps, and the fractions containing a product enriched in a given cannabinoid acid obtained from elution reverse phase step or steps are re-dissolved in an elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid acid such as tetrahydrocannabinolic acid (THCA), are dissolved in an elution solution and subjected to an ion exchange chromatographic purification step to produce substantially pure extracts. In alternative embodiments, the product of the ion exchange chromatographic step is collected in multiple fractions, which may then be tested for the presence of the desired cannabinoid or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid may then be selected for further purification. In alternative embodiments, the elution solution is removed from the selected fractions or optionally additional solution is added to change the polarity and/or hydrogen potential of the solution contained in the selected fraction. An exemplary embodiment comprises having the fractions enriched in a given cannabinoid obtained from the elution ion exchange chromatographic step mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. The solvent can then be removed.

In alternative embodiments, the selected fraction is then subjected to one or more additional ion exchange chromatographic steps and one or more isocratic or gradient elution solutions modifications. The ion exchange chromatographic step can comprise column chromatography in fixed or continuous mode, and can be based on molecular sizing and polarity ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™.

In alternative embodiments, various different elution solutions may be used in combination with this type of matrix, for example dimethyl sulfoxide, water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc.

In alternative embodiments, essential features of the process are the same for purification of all cannabinoids and cannabinoid acids. Cannabis plants generally contain complex mixtures of cannabinoid acids and cannabinoids, although depending on the variety of Cannabis one type of cannabinoid may pre-dominate. The purpose of the elution (e.g., gradient elution) chromatographic steps (ii) is to separate the various cannabinoid and/or cannabinoid/cannabinoid acid components of the crude plant extract loaded in step (i), as described above, into substantially pure fractions or substantially pure mixtures of fractions which are then optionally subjected to isomerization reactions and/or elimination reactions and/or additional gradient elution chromatography comprising normal phase, reverse phase and/or ion exchange.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which may then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid/cannabinoid acid may then be selected for further purification. Hence, the same simple process steps may be adapted for purification of essentially any plant-derived cannabinoid or cannabinoid acid. Selectivity for different cannabinoids or cannabinoid acids may be enhanced by selection of appropriate starting plant material. By way of example, if it is desired to prepare substantially pure Δ⁹ THC or Δ⁹ THCA then “high THC” Cannabis plants can be selected as the starting material. Alternatively, if it is desired to prepare substantially pure CBD or CBDA then “high CBD” Cannabis plants can be selected as the starting material. It is to be understood that the processes as provided herein are of general utility and are not limited to the use of particular Cannabis varieties as the starting material. The precise cannabinoid content of any particular Cannabis plant material can be qualitatively and quantitatively determined using analytical techniques well known to those skilled in the art, such as thin-layer chromatography or high-performance liquid chromatography (HPLC). Thus, one can screen a range of various Cannabis plants and select those having a high content of the desired cannabinoid acid or cannabinoid for use as starting material in a process as provided herein.

With the use of conventional selective breeding techniques it is possible to develop Cannabis varieties (chemovars) having varying cannabinoid content. Select Cannabis varieties (chemovars) have relatively high content of CBD, or of the minor cannabinoids Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), cannabigerol (CBG) or cannabichromene (CBC). General protocols for growing of medicinal Cannabis and for testing the cannabinoid content of Cannabis plants are described in International patent application WO 02/064109.

In alternative embodiments, methods further provide for the generation of a substantially pure preparation of Δ⁹ THC having a chromatographic purity of greater than about 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more by area normalization of an HPLC profile. The preparation can be a semi-solid at room temperature. The preparation can comprise less than about 1.5%, less than about 0.4%, or less than about 0.2%; or, less than about 0.1% CBD (w/w), less than about 0.5%, less than about 0.4%, or less than about 0.2%, or less than about 0.1% CBD (w/w) as analyzed by HPLC.

In alternative embodiments, the pure Δ⁸-THC and Δ⁹-THC provided by exemplary methods as provided herein have utility as an active pharmaceutical agent, and is also useful as a chromatographic standard, particularly as a comparative standard in the qualitative analysis of botanical drug substances derived from Cannabis. The availability of highly pure Δ⁸-THC and Δ⁹-THC will also facilitate studies of the pharmacology of Δ⁸-THC and Δ⁹-THC mixtures.

In alternative embodiments, an exemplary method for preparation of substantially pure Δ⁸-THC and Δ⁹ THC comprises:

-   -   i) obtaining an ethanolic solution of a botanical drug substance         from Cannabis or hemp plant material,     -   ii) passing the solution obtained in step i) through a filter,         and collecting the eluate,     -   iii) optionally substantially remove solvent from the eluate by         evaporation,     -   e.g., rotary evaporation, to give a cannabinoid enriched         fraction,     -   iv) optionally adding solvent to the eluate,     -   v) passing a solution of the resulting cannabinoid enriched         extract through a column packed with a stationary phase resin         such as DOWEX® MARATHON C Na™, MARATHON C H™, and conducting a         gradient elution of 50:1; 20:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1;         4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1         chloroform/methanol or ethanol,     -   vi) adjusting hydrogen potential of said solution,     -   vii) collecting Δ⁹-THC and CBD enriched fractions and optionally         removing solvent, e.g., by evaporation, e.g., rotary         evaporation,     -   viii) mixing the collected Δ⁹ THC and CBD with a reaction         solvent and a catalyst for a period of time and adding a         neutralizing agent and removing catalyst and neutralizing agent         by filtration to generate a reduced CBD and enriched Δ⁸-THC and         Δ⁹-THC mixture, optionally removing reaction solvent,     -   ix) mixing reduced CBD and enriched Δ⁸-THC and Δ⁹-THC mixture         with optionally additional reaction solvent and a stabilizing         agent, mixing stabilizing agent for a period of time     -   x) optionally removing the reaction solvent,     -   x) optionally re-dissolving the enriched Δ⁸ THC, Δ⁹ THC and         reduced CBD prepared in steps viii) through x) and passing the         solution through a column packed with reverse phase resin, and         conducting a gradient elution of 10:1; 9:1; 8:1; 7:1; 6:1; 5:1;         4:1; 3:1; 2:1; 1; 1 methanol/water,     -   xiii) optionally re-dissolving the crude Δ⁸ THC, Δ⁹ THC and         reduced CBD prepared in steps viii) through x) and passing the         solution through a column packed with an ion exchange resin such         as DOWEX® MARATHON C Na™, MARATHON C H™, and conducting an         elution of 50:1; 20:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1;         2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1 chloroform and polar solvent         mixture.     -   xiv) adjusting hydrogen potential of said solution     -   xv) collecting the THC enriched fractions and removing solvent,         e.g., by evaporation, e.g., rotary evaporation, to give a         semi-solid preparation of THC.

Example 2: Conversion of CBD to Δ⁸-THC and Δ⁹-THC

This Example describes an exemplary method for the conversion of CBD to Δ⁸-THC and Δ⁹-THC.

CBD (1 g) was added to 6.4 ml of reaction solvent and 1% p-toluenesulfonic acid. In this example, the mixture was reacted for 48 hours, although other time periods can also be used, as discussed below. It was then diluted with ether (20 ml) and poured into water, the upper layer was separated, washed with aqueous 5% NaHCO₃, then with water, dried over MgSO₄ and evaporated. GC-MS analysis on the crude product, showed the presence of 1.1% CBD; 18.4% Δ⁸-THC and 80.0% Δ⁹-THC. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The p-toluenesulfonic acid is used as a catalyst in the above example. It is of note that boron trifluoride could also be used as a catalyst, as could a number of other Lewis acids or non-oxidizing acid catalysts. The exact proportion is not essential to the reaction proceeding. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

In other embodiments, anhydrous MgSO₄ or another suitable agents such as Na₂SO₄, CaSO₄, and CaCl₂), known in the art is used in place of the MgSO₄.

Example 3: Conversion of CBD to Δ⁸-THC and Δ⁹-THC

This Example describes an exemplary method for the conversion of CBD to Δ⁸-THC and Δ⁹-THC.

A hemp extract was obtained by extraction of hemp in ethanol. The hemp extract was composed of CBD, THC, cannabinoids and other components and subjected to conversion of CBD to THC without further purification. The hemp extract and 1-5 mol % p-toluenesulfonic acid were sequentially added to 6.4 mL reaction solvent. In this example, the reaction mixture was stirred at room temperature for 24 hours, although other time periods can also be used, as discussed below. At some time intervals, the reaction mixture was diluted with ether (20 mL) and poured into water. The upper layer was separated, washed with aqueous 5% NaHCO₃, dried over MgSO4 and concentrated on a rotary evaporator. GC-MS analysis on the crude product showed the conversion of CBD to Δ⁸-THC and A-THC, for example, 0% CBD; 98% Δ⁹-THC; 2% Δ⁸-THC after 24 hours in the presence of 3 mol % p-toluenesulfonic acid. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The p-toluenesulfonic acid is used as a catalyst in the above example. Boron trifluoride could also be used as a catalyst, as could a number of other Lewis acids or non-oxidizing acids known in the art. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

In other embodiments, anhydrous MgSO₄ or another suitable agents such as Na₂SO₄, CaSO₄, and CaCl₂, known in the art is used in place of the MgSO₄.

Example 4: Exemplary Methods

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” is dissolved in a gradient elution solution and subjected to a chromatographic purification step to produce substantially pure cannabinoids and a product enriched in a given cannabinoid. The purpose of this step is to first remove the targeted non-cannabinoid material comprising for example terpenes, carotenes, and flavonoids and also to provide a degree of separation/fractionation of the various cannabinoid/cannabinoid acid components of the extract.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid and/or cannabinoid acid can then be selected for further purification. Optionally the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity or hydrogen potential of the gradient solvent contained in the selected fraction. The selected fraction can then be subjected to one or more additional chromatographic steps and one or more gradient elution solutions modifications.

In alternative embodiments, the chromatographic step comprises a column chromatography in a fixed or a continuous mode, and is can be based on molecular sizing, polarity and/or hydrogen potentiality. In alternative embodiments, the column matrix materials are, for example; silica (or silica gel) and alumina normal phase and reverse phase such as for example C18, C8, C4, C2, Amino, Cyano, Phenyl, Diol, WAX, SAX, WCX, SCX, Thiol; acidic, basic and neutral, styrenic, brominated styrenic; ion exchange resins such as strongly acidic, typically featuring sulfonic acid groups, e.g. sodium polystyrene sulfonate or polyAMPS; strongly basic, typically featuring quaternary amino groups, for example, trimethylammonium groups, e.g. polyAPTAC); weakly acidic, typically featuring carboxylic acid groups; weakly basic, typically featuring primary, secondary, and/or tertiary amino groups, e.g. polyethylene amine.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, hexane, butane, pentane, heptane, octane, carbon tetrachloride, etc.

In alternative embodiments, the chromatographic step comprises column chromatography on for example, a silica (or silica gel) or an alumina column, and the step can comprise eluting with a 5:1; 4:1; 3:1; 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1:1; 0.9:1; 0.5:1; 0.1:1 mixture of elution solutions such as chloroform, methylene dichloride, ethylene dichloride, methanol, ethanol, propanol and/or water. Any suitable combination of normal phase column packing material and solvent having separation characteristics suitable for use in separation (fractionation) of cannabinoids and/or cannabinoid acids can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. The fractions are tested for the presence of the desired cannabinoid and/or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid and/or cannabinoid acid selected for further processing. Solvent can be then removed from the selected fractions, e.g., by evaporation, e.g., rotary evaporation or equivalents.

In alternative embodiments, the fractions enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed.

In alternative embodiments, the fractions containing a product enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid such as mixtures comprising CBD and THC, or mixtures of CBD and CBG or mixtures of CBG and CBC, are dissolved in a gradient elution solution and subjected to a reverse phase chromatographic purification step to produce substantially pure extracts.

In alternative embodiments, the product of the reverse phase chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid and/or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity of the gradient solvent contained in the selected fraction.

An exemplary embodiment comprises: fractions enriched in a given cannabinoid obtained from the gradient elution reverse phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed. In alternative embodiments, the selected fraction is then subjected to one or more additional reverse phase chromatographic steps and one or more gradient elution solutions modifications. In alternative embodiments, the reverse phase chromatographic step comprises a column chromatography in fixed or continuous mode, and is can be based on polarity. Examples of reverse phase column chromatography include but are not limited to pure silica, alkyl chain-bonded silica, cyano-bonded silica, and phenyl-bonded silica.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, water, dimethylformamide, methanol, saline chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, dichloroethane, etc.

In alternative embodiments, the chromatographic step comprises column chromatography using an ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na™, Marathon C H™, Marathon MSC-1™ or Dowex Marathon WBA™ column, and eluting can be with a 20:1; 15:1; 10:1, 5:1; 4.5:1; 4:1; 3.5:1; 3:1 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1.001:1; 0.1:1 mixture of elution solutions such as chloroform, dichloromethane, dichloroethane methanol, ethanol, propanol, dimethyl sulfoxide, pyridine water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, dichloroethane, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc, and/or water. Any suitable combination of ion exchange phase column packing material and solvent having separation characteristics suitable for use in purification of cannabinoids and cannabinoids acid can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. In alternative embodiments, the fractions are tested for the presence of the desired cannabinoid or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid or cannabinoid acid. In alternative embodiments, solvent is then removed from the selected fractions, optionally by evaporation, e.g., rotary evaporation.

In alternative embodiments, the fractions containing a product are enriched in a given cannabinoid acid obtained from the gradient elution chromatographic step or steps, and the fractions containing a product enriched in a given cannabinoid acid obtained from gradient elution reverse phase step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid acid such as tetrahydrocannabinolic acid (THCA), are dissolved in a gradient elution solution and subjected to an ion exchange chromatographic purification step to produce substantially pure extracts. In alternative embodiments, the product of the ion exchange chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity and/or hydrogen potential of the gradient solvent contained in the selected fraction. An exemplary embodiment comprises: the fractions enriched in a given cannabinoid obtained from the gradient elution ion exchange chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. The solvent can then be removed.

In alternative embodiments, the selected fraction is then subjected to one or more additional ion exchange chromatographic steps and one or more gradient elution solutions modifications. The ion exchange chromatographic step can comprise column chromatography in fixed or continuous mode, and can be based on molecular sizing and polarity ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, water, dimethylformamide, methanol, ethylene dichloride, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, chloroform, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc.

In alternative embodiments, essential features of the process are the same for purification of all cannabinoids and cannabinoid acids. Cannabis plants generally contain complex mixtures of cannabinoid acids and cannabinoids, although depending on the variety of Cannabis one type of cannabinoid may pre-dominate. The purpose of the gradient elution chromatographic steps (ii) is to separate the various cannabinoid and/or cannabinoid/cannabinoid acid components of the crude plant extract loaded in step (i), as described above, into substantially pure fractions or substantially pure mixtures of fractions which are then optionally subjected to isomerization reactions and/or elimination reactions and/or additional gradient elution chromatography comprising normal phase, reverse phase and/or ion exchange.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid/cannabinoid acid can then be selected for further purification. Hence, the same simple process steps can be adapted for purification of essentially any plant-derived cannabinoid or cannabinoid acid. Selectivity for different cannabinoids or cannabinoid acids can be enhanced by selection of appropriate starting plant material. By way of example, if it is desired to prepare substantially pure Δ⁹ THC or Δ⁹ THCA then “high THC” Cannabis plants can be selected as the starting material. Alternatively, if it is desired to prepare substantially pure CBD or CBDA then “high CBD” Cannabis plants can be selected as the starting material. It is to be understood that processes as provided herein are of general utility and are not limited to the use of particular Cannabis varieties as the starting material. The precise cannabinoid content of any particular Cannabis plant material can be qualitatively and quantitatively determined using analytical techniques well known to those skilled in the art, such as thin-layer chromatography or high-performance liquid chromatography (HPLC). Thus, one may screen a range of various Cannabis plants and select those having a high content of the desired cannabinoid acid or cannabinoid for use as starting material in a process as provided herein.

With the use of conventional selective breeding techniques it is possible to develop Cannabis varieties (chemovars) having varying cannabinoid content. Select Cannabis varieties (chemovars) have relatively high content of CBD, or of the minor cannabinoids Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), cannabigerol (CBG) or cannabichromene (CBC). General protocols for growing of medicinal Cannabis and for testing the cannabinoid content of Cannabis plants are described in International patent application WO 02/064109.

In alternative embodiments, methods further provide for the generation of a substantially pure preparation of Δ⁹ THC having a chromatographic purity of greater than about 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more by area normalization of an HPLC profile. The preparation can be a semi-solid at room temperature. The preparation can comprises less than about 1.5%, less than about 0.4%, or less than about 0.2%; or, less than about 0.1% CBD (w/w), less than about 0.5%, less than about 0.4%, or less than about 0.2%, or less than about 0.1% CBD (w/w) as analyzed by HPLC.

In alternative embodiments, the pure Δ⁹-THC provided by exemplary methods as provided herein have utility as an active pharmaceutical agent, and is also useful as a chromatographic standard, particularly as a comparative standard in the qualitative analysis of botanical drug substances derived from Cannabis. The availability of highly pure Δ⁹-THC will also facilitate studies of the pharmacology of Δ⁹-THC.

In alternative embodiments, an exemplary method for preparation of substantially pure Δ⁹ THC comprises:

-   -   i) obtaining an ethanolic solution of a botanical drug substance         from Cannabis or hemp plant material,     -   ii) passing the solution obtained in step i) through a filter,         and collecting the eluate,     -   iii) optionally substantially remove solvent from the eluate,         e.g., by evaporation, e.g., rotary evaporation, to give a         cannabinoid enriched fraction,     -   iv) optionally adding solvent to the eluate,     -   v) passing a solution of the resulting cannabinoid enriched         extract through a column packed with a stationary phase resin         such as DOWEX® MARATHON C Na™, MARATHON C H™, and conducting a         gradient elution of 50:1; 20:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1;         4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1         chloroform/methanol or ethanol,     -   vi) adjusting hydrogen potential of said gradient solvent         solution,     -   vii) collecting Δ⁹-THC and CBD enriched fractions and optionally         removing solvent, e.g., by evaporation, e.g., rotary         evaporation,     -   viii) mixing the collected Δ⁹ THC and CBD with a reaction         solvent and a catalyst for a period of time and adding a         neutralizing agent and removing catalyst and neutralizing agent         by filtration to generate a CBD reduced Δ⁸-THC and Δ⁹-THC         mixture, optionally removing reaction solvent,     -   ix) mixing CBD reduced Δ⁸-THC and Δ⁹-THC mixture with optionally         additional reaction solvent and a stabilizing agent, mixing         stabilizing agent for a period of time,     -   x) mixing CBD reduced Δ⁸-THC and Δ⁹-THC and stabilizing agent         mixture with an elimination agent.     -   xi) separation of elimination agent and stabilizing agent from         substantially Δ⁹ THC,     -   xii) optionally re-dissolving the crude Δ⁹ THC and CBD prepared         in step xi) and passing the solution through a column packed         with reverse phase resin, and conducting a gradient elution of         10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1; 1         methanol/water,     -   xiii) optionally re-dissolving the crude Δ⁹ THC and CBD prepared         in step xi) such as DOWEX® MARATHON C Na™, MARATHON C H™, and         conducting a gradient elution of 50:1; 20:1; 10:1; 9:1; 8:1;         7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1         chloroform and polar solvent mixture.     -   vi) adjusting hydrogen potential of said gradient solvent         solution     -   vii) collecting the Δ⁹ THC enriched fractions and removing         solvent, e.g., by evaporation, e.g., rotary evaporation, to give         a semi-solid preparation of Δ⁹ THC.     -   (viii) collecting the CBD enriched fractions and removing         solvent, e.g., by evaporation, e.g., rotary evaporation, to give         a semi-solid preparation of CBD.

Example 5: Conversion of CBD to Δ⁸-THC and Δ⁹-THC

This Example describes an exemplary method for the conversion of CBD to Δ⁸-THC and Δ⁹-THC.

CBD (1 g) was added to 6.4 ml of reaction solvent and 1% p-toluenesulfonic acid. In this example, the mixture was reacted for 48 hours, although other time periods can also be used, as discussed below. It was then diluted with ether (20 ml) and poured into water, The upper layer was separated, washed with aqueous 5% NaHCO₃, then with water, dried over MgSO₄ and evaporated. GC-MS analysis on the crude product, showed the presence of 1.1% CBD; 18.4% Δ⁸-THC and 80.0% Δ⁹-THC. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The p-toluenesulfonic acid is used as a catalyst in the above example. It is of note that boron trifluoride could also be used as a catalyst, as could a number of other Lewis acids or non-oxidizing acid catalysts. The exact proportion is not essential to the reaction proceeding. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

In other embodiments, anhydrous MgSO₄ or another suitable agents such as Na₂SO₄, CaSO₄, and CaCl₂, known in the art is used in place of the MgSO₄.

Example 6: Conversion of Δ⁸ THC and Δ⁹ THC, and Purification of Δ⁹ THC

This Example describes an exemplary method for the conversion of Δ⁸-THC and Δ⁹-THC, and the purification of Δ⁹ THC.

A mixture containing 1.1% CBD; 18.4% Δ⁸-THC and 80.0% Δ⁹-THC (1 g) was added to 20 mL of reaction solvent and then 65 mol % protection agent was added to the solution. The colorless solution was saturated with hydrogen chloride at 0° C. in a dry apparatus and stirred. The mixture was washed with water and treated with sodium bicarbonate. The solvent layer was concentrated and dried under high vacuum. 3 equivalents of elimination agent was then added to the mixture in a dry solvent and heated up to 65° C. for 15 min. The mixture was mixed with solvent and washed with water and treated with sodium bicarbonate. The organic layer was dried over sodium sulfate and concentrated and dried under high vacuum. GC-MS analysis on the crude product, showed the presence of 1% CBD; 3% Δ⁸-THC and 96% Δ⁹-THC. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The zinc chloride is used as the protection agent in the above example. The exact proportion is not essential to the reaction proceeding. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

Example 7: Conversion of CBD to Δ⁸-THC and Δ⁹-THC

This Example describes an exemplary method for the conversion of CBD to Δ⁸-THC and Δ⁹-THC.

A hemp extract was obtained by extraction of hemp in ethanol. The hemp extract was composed of CBD, THC, cannabinoids and other components and subjected to conversion of CBD to THC without further purification. The hemp extract and 1-5 mol % p-toluenesulfonic acid were sequentially added to 6.4 mL reaction solvent. In this example, the reaction mixture was stirred at room temperature for 24 hours, although other time periods can also be used, as discussed below. At some time intervals, the reaction mixture was diluted with ether (20 mL) and poured into water. The upper layer was separated, washed with aqueous 5% NaHCO₃, dried over MgSO₄ and concentrated on a rotary evaporator. GC-MS analysis on the crude product showed the conversion of CBD to Δ⁸-THC and Δ⁸-THC, for example, 0% CBD; 98% Δ⁹-THC; 2% Δ⁸-THC after 24 hours in the presence of 3 mol % p-toluenesulfonic acid. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The p-toluenesulfonic acid is used as a catalyst in the above example. Boron trifluoride could also be used as a catalyst, as could a number of other Lewis acids or non-oxidizing acids known in the art. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

In other embodiments, anhydrous MgSO₄ or another suitable agents such as Na₂SO₄, CaSO₄, and CaCl₂), known in the art is used in place of the MgSO₄.

Example 8: Exemplary Methods

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” is dissolved in a gradient elution solution and subjected to a chromatographic purification step to produce substantially pure cannabinoids and a product enriched in a given cannabinoid. The purpose of this step is to first remove the targeted non-cannabinoid material comprising for example terpenes, carotenes, and flavonoids and also to provide a degree of separation/fractionation of the various cannabinoid/cannabinoid acid components of the extract.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which may then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid and/or cannabinoid acid can then be selected for further purification. Optionally the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity or hydrogen potential of the gradient solvent contained in the selected fraction. The selected fraction can then be subjected to one or more additional continuous chromatographic steps and one or more gradient elution solutions modifications.

In alternative embodiments, the chromatographic step comprises a column chromatography in a fixed or a continuous mode, and is can be based on molecular sizing, polarity and/or hydrogen potentiality. In alternative embodiments, the column matrix materials are, for example; silica (or silica gel) and alumina normal phase and reverse phase such as for example C18, C8, C4, C2, Amino, Cyano, Phenyl, Diol, WAX, SAX, WCX, SCX, Thiol; acidic, basic and neutral, styrenic, brominated styrenic; ion exchange resins such as strongly acidic, typically featuring sulfonic acid groups, e.g. sodium polystyrene sulfonate or polyAMPS; strongly basic, typically featuring quaternary amino groups, for example, trimethylammonium groups, e.g. polyAPTAC); weakly acidic, typically featuring carboxylic acid groups; weakly basic, typically featuring primary, secondary, and/or tertiary amino groups, e.g. polyethylene amine.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, hexane, butane, pentane, heptane, octane, carbon tetrachloride, etc.

In alternative embodiments, the chromatographic step comprises column chromatography on for example, a silica (or silica gel) or an alumina column, and the step can comprise eluting with a 5:1; 4:1; 3:1; 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1:1; 0.9:1; 0.5:1; 0.1:1 mixture of elution solutions such as chloroform, methylene dichloride, ethylene dichloride, methanol, ethanol, propanol and/or water. Any suitable combination of normal phase column packing material and solvent having separation characteristics suitable for use in separation (fractionation) of cannabinoids and/or cannabinoid acids can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. The fractions are tested for the presence of the desired cannabinoid and/or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid and/or cannabinoid acid selected for further processing. Solvent can be then removed from the selected fractions, e.g., by evaporation, e.g., rotary evaporation or equivalents.

In alternative embodiments, the fractions enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed.

In alternative embodiments, the fractions containing a product enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid such as mixtures comprising CBD and THC, or mixtures of CBD and CBG or mixtures of CBG and CBC, are dissolved in a gradient elution solution and subjected to a reverse phase chromatographic purification step to produce substantially pure extracts.

In alternative embodiments, the product of the reverse phase chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid and/or cannabinoid acid using any suitable analytical technique.

Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity of the gradient solvent contained in the selected fraction.

An exemplary embodiment comprises: fractions enriched in a given cannabinoid obtained from the gradient elution reverse phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed. In alternative embodiments, the selected fraction is then subjected to one or more additional reverse phase chromatographic steps and one or more gradient elution solutions modifications. In alternative embodiments, the reverse phase chromatographic step comprises a column chromatography in fixed or continuous mode, and is can be based on polarity. Examples of reverse phase column chromatography include but are not limited to pure silica, alkyl chain-bonded silica, cyano-bonded silica, and phenyl-bonded silica.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine water, dimethylformamide, methanol, saline chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, dichloroethane, etc.

In alternative embodiments, the chromatographic step comprises column chromatography using an ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™ column, and eluting can be with a 20:1; 15:1; 10:1, 5:1; 4.5:1; 4:1; 3.5:1; 3:1 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1.001:1; 0.1:1 mixture of elution solutions such as chloroform, dichloromethane, dichloroethane, methanol, ethanol, propanol, dimethyl sulfoxide, pyridine water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, chloroform dichloromethane, dichloroethane, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc, and/or water. Any suitable combination of ion exchange phase column packing material and solvent having separation characteristics suitable for use in purification of cannabinoids and cannabinoids acid can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. In alternative embodiments, the fractions are tested for the presence of the desired cannabinoid or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid or cannabinoid acid. In alternative embodiments, solvent is then removed from the selected fractions, optionally by evaporation, e.g., rotary evaporation.

In alternative embodiments, the fractions containing a product are enriched in a given cannabinoid acid obtained from the gradient elution chromatographic step or steps, and the fractions containing a product enriched in a given cannabinoid acid obtained from gradient elution reverse phase step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid acid such as tetrahydrocannabinolic acid (THCA), are dissolved in a gradient elution solution and subjected to an ion exchange chromatographic purification step to produce substantially pure extracts. In alternative embodiments, the product of the ion exchange chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity and/or hydrogen potential of the gradient solvent contained in the selected fraction. An exemplary embodiment comprises: the fractions enriched in a given cannabinoid obtained from the gradient elution ion exchange chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. The solvent can then be removed.

In alternative embodiments, the selected fraction is then subjected to one or more additional ion exchange chromatographic steps and one or more gradient elution solutions modifications. The ion exchange chromatographic step can comprise column chromatography in fixed or continuous mode, and can be based on molecular sizing and polarity ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc.

In alternative embodiments, essential features of the process are the same for purification of all cannabinoids and cannabinoid acids. Cannabis plants generally contain complex mixtures of cannabinoid acids and cannabinoids, although depending on the variety of Cannabis one type of cannabinoid may pre-dominate. The purpose of the gradient elution chromatographic steps (ii) is to separate the various cannabinoid and/or cannabinoid/cannabinoid acid components of the crude plant extract loaded in step (i), as described above, into substantially pure fractions or substantially pure mixtures of fractions which are then optionally subjected to isomerization reactions and/or elimination reactions and/or additional gradient elution chromatography comprising normal phase, reverse phase and/or ion exchange.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid/cannabinoid acid can then be selected for further purification. Hence, the same simple process steps can be adapted for purification of essentially any plant-derived cannabinoid or cannabinoid acid. Selectivity for different cannabinoids or cannabinoid acids can be enhanced by selection of appropriate starting plant material. By way of example, if it is desired to prepare substantially pure Δ⁸ THC then high CBD and/or high Δ⁹ THC and/or Δ⁹ THCA Cannabis plants can be selected as the starting material. Alternatively, if it is desired to prepare substantially pure CBD or CBDA then “high CBD” Cannabis plants can be selected as the starting material. I is to be understood that processes as provided herein are of general utility and are not limited to the use of particular Cannabis varieties as the starting material. The precise cannabinoid content of any particular Cannabis plant material can be qualitatively and quantitatively determined using analytical techniques well known to those skilled in the art, such as thin-layer chromatography or high-performance liquid chromatography (HPLC). Thus, one can screen a range of various Cannabis plants and select those having a high content of the desired cannabinoid acid or cannabinoid for use as starting material in a process as provided herein.

With the use of conventional selective breeding techniques it is possible to develop Cannabis varieties (chemovars) having varying cannabinoid content. Select Cannabis varieties (chemovars) have relatively high content of CBD, or of the minor cannabinoids Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), cannabigerol (CBG) or cannabichromene (CBC). General protocols for growing of medicinal Cannabis and for testing the cannabinoid content of Cannabis plants are described in International patent application WO 02/064109.

In alternative embodiments, methods further provide for the generation of a substantially pure preparation of Δ⁸ THC having a chromatographic purity of greater than about 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more by area normalization of an HPLC profile. The preparation can be a semi-solid at room temperature. The preparation can comprises less than about 1.5%, less than about 0.4%, or less than about 0.2%; or, less than about 0.1% CBD (w/w), less than about 0.5%, less than about 0.4%, or less than about 0.2%, or less than about 0.1% CBD (w/w) as analyzed by HPLC.

In alternative embodiments, the pure Δ⁸-THC provided by exemplary methods as provided herein have utility as an active pharmaceutical agent, and is also useful as a chromatographic standard, particularly as a comparative standard in the qualitative analysis of botanical drug substances derived from Cannabis. The availability of highly pure Δ⁸-THC will also facilitate studies of the pharmacology of Δ⁸-THC.

In alternative embodiments, an exemplary method for preparation of substantially pure Δ⁹ THC comprises:

-   -   i) obtaining an ethanolic solution of a botanical drug substance         from Cannabis or hemp plant material,     -   ii) passing the solution obtained in step i) through a filter,         and collecting the eluate,     -   iii) optionally substantially remove solvent from the eluate,         e.g., by evaporation, e.g., rotary evaporation, to give a         cannabinoid enriched fraction,     -   iv) optionally adding solvent to the eluate,     -   v) passing a solution of the resulting cannabinoid enriched         extract through a column packed with a stationary phase resin         such as DOWEX® MARATHON C Na™, MARATHON C H™, and conducting a         gradient elution of 50:1; 20:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1;         4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1         chloroform/methanol or ethanol,     -   vi) adjusting hydrogen potential of said gradient solvent         solution,     -   vii) collecting Δ⁸-THC, Δ⁹-THC and CBD enriched fractions and         optionally removing solvent, e.g., by evaporation, e.g., rotary         evaporation,     -   viii) mixing the collected Δ⁸-THC, Δ⁹ THC and CBD with a         reaction solvent and a catalyst for a period of time and adding         a neutralizing agent and removing catalyst and neutralizing         agent by filtration to generate a substantially pure Δ⁸-THC         product with a reduced CBD and Δ⁹-THC content, optionally         removing reaction solvent,     -   viii) optionally re-dissolving the substantially Δ⁸ THC mixture         prepared in step viii) and passing the solution through a column         packed with reverse phase resin, and conducting a gradient         elution of 10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1; 1         methanol/water,     -   x) optionally re-dissolving the substantially Δ⁸ THC prepared in         step viii) such as DOWEX® MARATHON C Na™, MARATHON C H™, and         conducting a gradient elution of 50:1; 20:1; 10:1; 9:1; 8:1;         7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1         chloroform and polar solvent mixture.     -   xi) adjusting hydrogen potential of said gradient solvent         solution     -   xii) collecting the Δ⁸ THC enriched fractions and removing         solvent, e.g., by evaporation, e.g., rotary evaporation to give         a semi-solid preparation of Δ⁸ THC.

Example 9: Conversion of CBD to Δ⁸-THC and Δ⁹-THC

This Example describes an exemplary method for the conversion of CBD to Δ⁸-THC.

CBD (15 g) was added to 240 ml of reaction solvent and p-toluenesulfonic acid. In this example, the mixture was reacted for 27 hours, although other time periods can also be used, as discussed below. It was then diluted with ether (20 ml) and poured into water. The upper layer was separated, washed with aqueous 5% NaHCO₃, then with water, dried over MgSO₄ and evaporated. GC-MS analysis on the crude product, showed the presence of 96.5% Δ⁸-THC and 2.5% Δ⁹-THC. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The p-toluenesulfonic acid is used as a catalyst in the above example. It is of note that boron trifluoride could also be used as a catalyst, as could a number of other Lewis acids or non-oxidizing acid catalysts. The exact proportion is not essential to the reaction proceeding. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

In other embodiments, anhydrous MgSO₄ or another suitable agents such as Na₂SO₄, CaSO₄, and CaCl₂), known in the art is used in place of the MgSO₄.

Example 10: Exemplary Methods

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” is dissolved in a gradient elution solution and subjected to one or multiple continuous chromatographic purification step to produce substantially pure cannabinoids and a product enriched in a given cannabinoid. The purpose of this step is to first remove the targeted non-cannabinoid material comprising for example terpenes, carotenes, and flavonoids and also to provide a degree of separation/fractionation of the various cannabinoid/cannabinoid acid components of the extract.

In alternative embodiments, the product of one or more continuous chromatographic steps is collected in multiple fractions, which may then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid and/or cannabinoid acid can then be selected for further purification. Optionally the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity or hydrogen potential of the gradient solvent contained in the selected fraction. The selected fraction can then be subjected to one or more additional chromatographic steps and one or more gradient elution solutions modifications.

In alternative embodiments, the chromatographic step comprises a column chromatography in a continuous mode, and is based on molecular sizing, polarity and/or hydrogen potentiality. In alternative embodiments, the column matrix materials are, for example; silica (or silica gel), alumina and reverse phase for example C18, C8, C4, C2, Amino, Cyano, Phenyl, Diol, WAX, SAX, WCX, SCX, Thiol; acidic, basic and neutral, styrenic, brominated styrenic; ion exchange resins such as strongly acidic, typically featuring sulfonic acid groups, e.g. sodium polystyrene sulfonate or polyAMPS; strongly basic, typically featuring quaternary amino groups, for example, trimethylammonium groups, e.g. polyAPTAC); weakly acidic, typically featuring carboxylic acid groups; weakly basic, typically featuring primary, secondary, and/or tertiary amino groups, e.g. polyethylene amine.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine, water, dimethylformamide, methanol, saline, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, hexane, butane, pentane, heptane, octane, carbon tetrachloride, etc.

In alternative embodiments, the chromatographic step comprises continuous column chromatography on for example, a silica (or silica gel) or an alumina column, and the step can comprise eluting with a 5:1; 4:1; 3:1; 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1:1; 0.9:1; 0.5:1; 0.1:1 mixture of elution solutions such as chloroform, methylene dichloride, ethylene dichloride, methanol, ethanol, propanol and/or water. Any suitable combination of normal phase column packing material and solvent having separation characteristics suitable for use in separation (fractionation) of cannabinoids and/or cannabinoid acids can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. The fractions are tested for the presence of the desired cannabinoid and/or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid and/or cannabinoid acid selected for further processing. Solvent can be then removed from the selected fractions, e.g., by evaporation, e.g., rotary evaporation or equivalents.

In alternative embodiments, the fractions enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed.

In alternative embodiments, the fractions containing a product enriched in a given cannabinoid obtained from the gradient elution normal phase continuous chromatographic step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid such as mixtures comprising CBD and THC, or mixtures of CBD and CBG or mixtures of CBG and CBC, are dissolved in a gradient elution solution and subjected to a continuous reverse phase chromatographic purification step to produce substantially pure extracts.

In alternative embodiments, the product of the reverse phase chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid and/or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity of the gradient solvent contained in the selected fraction.

An exemplary embodiment comprises: fractions enriched in a given cannabinoid obtained from the gradient elution reverse phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed. In alternative embodiments, the selected fraction is then subjected to one or more additional reverse phase chromatographic steps and one or more gradient elution solutions modifications. In alternative embodiments, the reverse phase chromatographic step comprises a column chromatography continuous mode, and can be based on polarity or affinity.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine water, dimethylformamide, methanol, saline chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, dichloroethane, etc.

In alternative embodiments, the chromatographic step comprises continuous column chromatography using an ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™ column, and eluting can be with a 20:1; 15:1; 10:1, 5:1; 4.5:1; 4:1; 3.5:1; 3:1 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1.001:1; 0.1:1 mixture of elution solutions such as chloroform, dichloromethane, dichloroethane methanol, ethanol, propanol, dimethyl sulfoxide, water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, chloroform dichloromethane, dichloroethane, acetic acid, carbonic acid, glycolic acid, benzoic, methanoic, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc, and/or water. Any suitable combination of ion exchange phase column packing material and solvent having separation characteristics suitable for use in purification of cannabinoids and cannabinoids acid can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. In alternative embodiments, the fractions are tested for the presence of the desired cannabinoid or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid or cannabinoid acid. In alternative embodiments, solvent is then removed from the selected fractions, optionally by evaporation, e.g., rotary evaporation.

In alternative embodiments, the fractions containing a product are enriched in a given cannabinoid acid obtained from the continuous gradient elution chromatographic step or steps, and the fractions containing a product enriched in a given cannabinoid acid obtained from gradient elution reverse phase step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid acid such as tetra-hydrocannabinolic acid (THCA), are dissolved in a gradient elution solution and subjected to a continuous ion exchange chromatographic purification step to produce substantially pure extracts. In alternative embodiments, the product of the ion exchange chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity and/or hydrogen potential of the gradient solvent contained in the selected fraction. An exemplary embodiment comprises: the fractions enriched in a given cannabinoid obtained from the gradient elution ion exchange chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. The solvent can then be removed.

In alternative embodiments, the selected fraction is then subjected to one or more additional continuous ion exchange chromatographic steps and one or more gradient elution solutions modifications. The continuous ion exchange chromatographic step can comprise column chromatography in fixed or continuous mode, and can be based on molecular sizing and polarity ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine, water, dimethylformamide, methanol, saline, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, chloroform, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc.

In alternative embodiments, essential features of the process are the same for purification of all cannabinoids and cannabinoid acids. Cannabis plants generally contain complex mixtures of cannabinoid acids and cannabinoids, although depending on the variety of Cannabis one type of cannabinoid may pre-dominate. The purpose of the gradient elution chromatographic steps (ii) is to continuously separate the various cannabinoid and/or cannabinoid/cannabinoid acid components of the crude plant extract loaded in step (i), as described above, into substantially pure fractions or substantially pure mixtures of fractions which are then optionally subjected to additional gradient elution chromatography comprising normal phase, reverse phase and/or ion exchange.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid/cannabinoid acid can then be selected for further purification. Hence, the same simple process steps can be adapted for purification of essentially any plant-derived cannabinoid or cannabinoid acid. Selectivity for different cannabinoids or cannabinoid acids can be enhanced by selection of appropriate starting plant material. By way of example, if it is desired to prepare substantially pure Δ⁹ THC or Δ⁹ THCA then “high THC” Cannabis plants can be selected as the starting material. Alternatively, if it is desired to prepare substantially pure CBD or CBDA then “high CBD” Cannabis plants can be selected as the starting material. It is to be understood that processes are provided herein are of general utility and are not limited to the use of particular Cannabis varieties as the starting material. The precise cannabinoid content of any particular Cannabis plant material can be qualitatively and quantitatively determined using analytical techniques well known to those skilled in the art, such as thin-layer chromatography or high-performance liquid chromatography (HPLC). Thus, one may screen a range of various Cannabis plants and select those having a high content of the desired cannabinoid acid or cannabinoid for use as starting material in a process as provided herein.

With the use of conventional selective breeding techniques it is possible to develop Cannabis varieties (chemovars) having varying cannabinoid content. Select Cannabis varieties (chemovars) have relatively high content of CBD, or of the minor cannabinoids Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), cannabigerol (CBG) or cannabichromene (CBC). General protocols for growing of medicinal Cannabis and for testing the cannabinoid content of Cannabis plants are described in International patent application WO 02/064109.

In alternative embodiments, methods further provide for the generation of a substantially pure preparation of Δ⁹ THC having a chromatographic purity of greater than about 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more by area normalization of an HPLC profile. The preparation can be a semi-solid at room temperature. The preparation can comprises less than about 1.5%, less than about 0.4%, or less than about 0.2%; or, less than about 0.1% CBD (w/w), less than about 0.5%, less than about 0.4%, or less than about 0.2%, or less than about 0.1% CBD (w/w) as analyzed by HPLC.

In alternative embodiments, the pure Δ⁹-THC provided by exemplary methods as provided herein have utility as an active pharmaceutical agent, and is also useful as a chromatographic standard, particularly as a comparative standard in the qualitative analysis of botanical drug substances derived from Cannabis. The availability of highly pure Δ⁹-THC will also facilitate studies of the pharmacology of Δ⁹-THC.

Exemplary Method

-   -   i) obtaining an ethanolic solution of a botanical drug substance         from Cannabis or hemp plant material,     -   ii) passing the solution obtained in step i) through a filter,         and collecting the eluate,     -   iii) optionally substantially remove solvent from the eluate,         e.g., by evaporation, e.g., rotary evaporation to give a         cannabinoid enriched fraction,     -   iv) optionally adding solvent to the eluate,     -   v) continuously loading a solution of the resulting cannabinoid         enriched extract onto a column packed with a stationary phase         resin such as DOWEX® MARATHON C Na™, MARATHON C H™, and         continuously conducting a gradient elution of 50:1; 20:1; 10:1;         9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1;         0.0005:1 chloroform/methanol or ethanol,     -   vi) adjusting hydrogen potential of said gradient solvent         solution,     -   vii) collecting Δ⁹-THC and CBD enriched fractions and optionally         removing solvent, e.g., by evaporation, e.g., rotary         evaporation,     -   viii) optionally re-dissolving the crude Δ⁹ THC and CBD prepared         in step vii) and continuously loading the solution on a column         packed with a normal phase reverse phase resin, and continuously         conducting a gradient elution of 10:1; 9:1; 8:1; 7:1; 6:1; 5:1;         4:1; 3:1; 2:1; 1; 1 methanol/water,     -   xiii) optionally re-dissolving the crude Δ⁹ THC and CBD prepared         in step vii) such as DOWEX® MARATHON C Na™, MARATHON C H™, and         conducting a gradient elution of 50:1; 20:1; 10:1; 9:1; 8:1;         7:1; 6:1; 5:1; 4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1         chloroform and polar solvent mixture.     -   vi) adjusting hydrogen potential of said gradient solvent         solution     -   vii) collecting the Δ⁹ THC enriched fractions and removing         solvent, e.g., by evaporation, e.g., rotary evaporation to give         a semi-solid preparation of Δ⁹ THC.     -   (viii) collecting the CBD enriched fractions and removing         solvent, e.g., by evaporation, e.g., rotary evaporation to give         a semi-solid preparation of CBD.

Example 11: Exemplary Methods

In alternative embodiments, the “extract containing a cannabinoid or a cannabinoid acid” is dissolved in a gradient elution solution and subjected to a chromatographic purification step to produce substantially pure cannabinoids and a product enriched in a given cannabinoid. The purpose of this step is to first remove the targeted non-cannabinoid material comprising for example terpenes, carotenes, and flavonoids and also to provide a degree of separation/fractionation of the various cannabinoid/cannabinoid acid components of the extract.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid and/or cannabinoid acid can then be selected for further purification. Optionally the gradient elution solution is removed from the selected fractions or optionally additional gradient solvent is added to change the polarity or hydrogen potential of the gradient solvent contained in the selected fraction. The selected fraction can then be subjected to one or more additional chromatographic steps and one or more gradient elution solutions modifications.

In alternative embodiments, the chromatographic step comprises a column chromatography in a fixed or a continuous mode, and is can be based on molecular sizing, polarity and/or hydrogen potentiality. In alternative embodiments, the column matrix materials are, for example; silica (or silica gel) and alumina normal phase and reverse phase such as for example C18, C8, C4, C2, Amino, Cyano, Phenyl, Diol, WAX, SAX, WCX, SCX, Thiol; acidic, basic and neutral, styrenic, brominated styrenic; ion exchange resins such as strongly acidic, typically featuring sulfonic acid groups, e.g. sodium polystyrene sulfonate or polyAMPS; strongly basic, typically featuring quaternary amino groups, for example, trimethylammonium groups, e.g. polyAPTAC); weakly acidic, typically featuring carboxylic acid groups; weakly basic, typically featuring primary, secondary, and/or tertiary amino groups, e.g. polyethylene amine.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine, water, dimethylformamide, methanol, saline, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, hexane, butane, pentane, heptane, octane, carbon tetrachloride, etc.

In alternative embodiments, the chromatographic step comprises column chromatography on for example, a silica (or silica gel) or an alumina column, and the step can comprise eluting with a 5:1; 4:1; 3:1; 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1:1; 0.9:1; 0.5:1; 0.1:1 mixture of elution solutions such as chloroform, methylene dichloride, ethylene dichloride, methanol, ethanol, propanol and/or water. Any suitable combination of normal phase column packing material and solvent having separation characteristics suitable for use in separation (fractionation) of cannabinoids and/or cannabinoid acids can be used with equivalent effect.

In alternative embodiments, the column gradient eluate is collected in several fractions. The fractions are tested for the presence of the desired cannabinoid and/or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid and/or cannabinoid acid selected for further processing. Solvent can be then removed from the selected fractions, e.g., by rotary evaporation or equivalents.

In alternative embodiments, the fractions enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed.

In alternative embodiments, the fractions containing a product enriched in a given cannabinoid obtained from the gradient elution normal phase chromatographic step or steps are re-dissolved in a gradient elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid such as mixtures comprising CBD and THC, or mixtures of CBD and CBG or mixtures of CBG and CBC, are dissolved in a gradient elution solution and subjected to a reverse phase chromatographic purification step to produce substantially pure extracts.

In alternative embodiments, the product of the reverse phase chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid and/or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the elution solution is removed from the selected fractions or optionally additional solvent is added to change the polarity of the solution contained in the selected fraction.

An exemplary embodiment comprises: fractions enriched in a given cannabinoid obtained from the elution of a reverse phase chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. In alternative embodiments, the solvent is then removed. In alternative embodiments, the selected fraction is then subjected to one or more additional reverse phase chromatographic steps and one or more elution solutions modifications. In alternative embodiments, the reverse phase chromatographic step comprises a column chromatography in fixed or continuous mode, and can be based on polarity. Examples of reverse phase column chromatography include but are not limited to pure silica, alkyl chain-bonded silica, cyano-bonded silica, and phenyl-bonded silica.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, pyridine, water, dimethylformamide, methanol, saline, chloroform, propanol, ethanol, isobutanol, formamide, butanol, isopropanol, tetrahydrofuran, dioxane, chloroform, dichloromethane, dichloroethane, etc.

In alternative embodiments, the chromatographic step comprises column chromatography using an ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™ column, and eluting can be with a 20:1; 15:1; 10:1, 5:1; 4.5:1; 4:1; 3.5:1; 3:1 2:1; 1.9:1; 1.8:1; 1.7:1; 1.6:1; 1.5:1; 1.4:1; 1.3:1; 1.2:1; 1.1:1; 1.01:1; 1.001:1; 0.1:1 mixture of elution solutions such as chloroform, dichloromethane, dichloroethane methanol, ethanol, propanol, dimethyl sulfoxide, water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, dichloromethane, dichloroethane, acetic acid, carbonic acid, glycolic acid, benzoic, methanoic, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc, and/or water. Any suitable combination of ion exchange phase column packing material and solvent having separation characteristics suitable for use in purification of cannabinoids and cannabinoids acid can be used with equivalent effect.

In alternative embodiments, the column eluate is collected in several fractions. In alternative embodiments, the fractions are tested for the presence of the desired cannabinoid or cannabinoid acid using a suitable analytical technique, and those fractions containing the highest amounts of the desired cannabinoid or cannabinoid acid. In alternative embodiments, solvent is then removed from the selected fractions, optionally by rotary evaporation.

In alternative embodiments, the fractions containing a product are enriched in a given cannabinoid acid obtained from the elution chromatographic step or steps, and the fractions containing a product enriched in a given cannabinoid acid obtained from elution reverse phase step or steps are re-dissolved in an elution solution. In alternative embodiments, the fractions containing a product enriched in a given cannabinoid acid such as tetrahydrocannabinolic acid (THCA), are dissolved in an elution solution and subjected to an ion exchange chromatographic purification step to produce substantially pure extracts. In alternative embodiments, the product of the ion exchange chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid or cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid or cannabinoid acid can then be selected for further purification. In alternative embodiments, the elution solution is removed from the selected fractions or optionally additional solution is added to change the polarity and/or hydrogen potential of the solution contained in the selected fraction. An exemplary embodiment comprises: the fractions enriched in a given cannabinoid obtained from the elution ion exchange chromatographic step are mixed with a solvent of the opposite polarity thereby causing a concentration of the cannabinoids in the second solvent phase. The solvent can then be removed.

In alternative embodiments, the selected fraction is then subjected to one or more additional ion exchange chromatographic steps and one or more isocratic or gradient elution solutions modifications. The ion exchange chromatographic step can comprise column chromatography in fixed or continuous mode, and can be based on molecular sizing and polarity ion exchange resin such as but not limited to RediSep® Rf SAX, Dowex® Marathon C Na, Marathon C H, Marathon MSC-1 or Dowex Marathon WBA™.

In alternative embodiments, various different elution solutions can be used in combination with this type of matrix, for example dimethyl sulfoxide, water, dimethylformamide, methanol, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, isopropanol, tetrahydrofuran, dioxane, acetic acid, carbonic acid, glycolic acid, benzoic acid, formic acid, oxalic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, ammonium carbonate, etc.

In alternative embodiments, essential features of the process are the same for purification of all cannabinoids and cannabinoid acids. Cannabis plants generally contain complex mixtures of cannabinoid acids and cannabinoids, although depending on the variety of Cannabis one type of cannabinoid may pre-dominate. The purpose of the elution (e.g., gradient elution) chromatographic steps (ii) is to separate the various cannabinoid and/or cannabinoid/cannabinoid acid components of the crude plant extract loaded in step (i), as described above, into substantially pure fractions or substantially pure mixtures of fractions which are then optionally subjected to isomerization reactions and/or elimination reactions and/or additional gradient elution chromatography comprising normal phase, reverse phase and/or ion exchange.

In alternative embodiments, the product of the chromatographic step is collected in multiple fractions, which can then be tested for the presence of the desired cannabinoid/cannabinoid acid using any suitable analytical technique. Fractions enriched in the desired cannabinoid/cannabinoid acid can then be selected for further purification. Hence, the same simple process steps can be adapted for purification of essentially any plant-derived cannabinoid or cannabinoid acid. Selectivity for different cannabinoids or cannabinoid acids may be enhanced by selection of appropriate starting plant material. By way of example, if it is desired to prepare substantially pure Δ⁹ THC or Δ⁹ THCA then “high THC” Cannabis plants can be selected as the starting material. Alternatively, if it is desired to prepare substantially pure CBD or CBDA then “high CBD” Cannabis plants can be selected as the starting material. It is to be understood that processes as provided herein are of general utility and are not limited to the use of particular Cannabis varieties as the starting material. The precise cannabinoid content of any particular Cannabis plant material can be qualitatively and quantitatively determined using analytical techniques well known to those skilled in the art, such as thin-layer chromatography or high-performance liquid chromatography (HPLC). Thus, one may screen a range of various Cannabis plants and select those having a high content of the desired cannabinoid acid or cannabinoid for use as starting material in a process as provided herein.

With the use of conventional selective breeding techniques it is possible to develop Cannabis varieties (chemovars) having varying cannabinoid content. Select Cannabis varieties (chemovars) have relatively high content of CBD, or of the minor cannabinoids Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), cannabigerol (CBG) or cannabichromene (CBC). General protocols for growing of medicinal Cannabis and for testing the cannabinoid content of Cannabis plants are described in International patent application WO 02/064109.

In alternative embodiments, methods further provide for the generation of a substantially pure preparation of Δ⁹ THC having a chromatographic purity of greater than about 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more by area normalization of an HPLC profile. The preparation can be a semi-solid at room temperature. The preparation can comprises less than about 1.5%, less than about 0.4%, or less than about 0.2%; or, less than about 0.1% CBD (w/w), less than about 0.5%, less than about 0.4%, or less than about 0.2%, or less than about 0.1% CBD (w/w) as analyzed by HPLC.

In alternative embodiments, the pure Δ⁸-THC and Δ⁹-THC provided by exemplary methods as provided herein have utility as an active pharmaceutical agent, and is also useful as a chromatographic standard, particularly as a comparative standard in the qualitative analysis of botanical drug substances derived from Cannabis. The availability of highly pure Δ⁸-THC and Δ⁹-THC will also facilitate studies of the pharmacology of Δ⁸-THC and Δ⁹-THC mixtures.

In alternative embodiments, an exemplary method for preparation of substantially pure Δ⁸-THC and Δ⁹ THC comprises:

-   -   i) obtaining an ethanolic solution of a botanical drug substance         from Cannabis or hemp plant material,     -   ii) passing the solution obtained in step i) through a filter,         and collecting the eluate,     -   iii) optionally substantially remove solvent from the eluate by         rotary evaporation to give a cannabinoid enriched fraction,     -   iv) optionally adding solvent to the eluate,     -   v) passing a solution of the resulting cannabinoid enriched         extract through a column packed with a stationary phase resin         such as DOWEX® MARATHON C Na™, MARATHON C H™, and conducting a         gradient elution of 50:1; 20:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1;         4:1; 3:1; 2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1         chloroform/methanol or ethanol,     -   vi) adjusting hydrogen potential of said solution,     -   vii) collecting Δ⁹-THC and CBD enriched fractions and optionally         removing solvent by rotary evaporation,     -   viii) mixing the collected Δ⁹ THC and CBD with a reaction         solvent and a catalyst for a period of time and adding a         neutralizing agent and removing catalyst and neutralizing agent         by filtration to generate a reduced CBD and enriched Δ⁸-THC and         Δ⁹-THC mixture, optionally removing reaction solvent,     -   ix) mixing reduced CBD and enriched Δ⁸-THC and Δ⁹-THC mixture         with optionally additional reaction solvent and a stabilizing         agent, mixing stabilizing agent for a period of time     -   x) optionally removing the reaction solvent,     -   x) optionally re-dissolving the enriched Δ⁸ THC, Δ⁹ THC and         reduced CBD prepared in steps viii) through x) and passing the         solution through a column packed with reverse phase resin, and         conducting a gradient elution of 10:1; 9:1; 8:1; 7:1; 6:1; 5:1;         4:1; 3:1; 2:1; 1; 1 methanol/water,     -   xiii) optionally re-dissolving the crude Δ⁸ THC, Δ⁹ THC and         reduced CBD prepared in steps viii) through x) and passing the         solution through a column packed with an ion exchange resin such         as DOWEX® MARATHON C Na™, MARATHON C H™, and conducting an         elution of 50:1; 20:1; 10:1; 9:1; 8:1; 7:1; 6:1; 5:1; 4:1; 3:1;         2:1; 1; 1; 0.5:1; 0.005:1; 0.0005:1 chloroform and polar solvent         mixture.     -   xiv) adjusting hydrogen potential of said solution     -   xv) collecting the THC enriched fractions and removing solvent         by rotary evaporation to give a semi-solid preparation of THC.

Example 12: Conversion of CBD to Δ⁸-THC and Δ⁹-THC

This Example describes an exemplary method for the conversion of CBD to Δ⁸-THC and Δ⁹-THC.

CBD (1 g) was added to 6.4 ml of reaction solvent and 1% p-toluenesulfonic acid. In this example, the mixture was reacted for 48 hours, although other time periods can also be used, as discussed below. It was then diluted with ether (20 ml) and poured into water, The upper layer was separated, washed with aqueous 5% NaHCO₃, then with water, dried over MgSO₄ and evaporated. GC-MS analysis on the crude product, showed the presence of 1.1% CBD; 18.4% Δ⁸-THC and 80.0% Δ⁹-THC. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The p-toluenesulfonic acid is used as a catalyst in the above example. It is of note that boron trifluoride could also be used as a catalyst, as could a number of other Lewis acids or non-oxidizing acid catalysts. The exact proportion is not essential to the reaction proceeding. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

In other embodiments, anhydrous MgSO₄ or another suitable agents such as Na₂SO₄, CaSO₄, and CaCl₂, known in the art is used in place of the MgSO₄.

Example 13: Conversion of CBD to Δ⁸-THC and Δ⁹-THC

This Example describes an exemplary method for the conversion of CBD to Δ⁸-THC and Δ⁹-THC.

A hemp extract was obtained by extraction of hemp in ethanol. The hemp extract was composed of CBD, THC, cannabinoids and other components and subjected to conversion of CBD to THC without further purification. The hemp extract and 1-5 mol % p-toluenesulfonic acid were sequentially added to 6.4 mL reaction solvent. In this example, the reaction mixture was stirred at room temperature for 24 hours, although other time periods can also be used, as discussed below. At some time intervals, the reaction mixture was diluted with ether (20 mL) and poured into water. The upper layer was separated, washed with aqueous 5% NaHCO₃, dried over MgSO4 and concentrated on a rotary evaporator. GC-MS analysis on the crude product showed the conversion of CBD to Δ⁸-THC and A-THC, for example, 0% CBD; 98% Δ⁹-THC; 2% Δ⁸-THC after 24 hours in the presence of 3 mol % p-toluenesulfonic acid. The crude product was then subjected to column chromatography. In the example described above, normal phase HPLC separation is used wherein the column is for example a silica gel and the mobile phase is an organic solvent mixture introduced as a gradient described in the invention. In other embodiments, reverse phase HPLC separation is used.

The p-toluenesulfonic acid is used as a catalyst in the above example. Boron trifluoride could also be used as a catalyst, as could a number of other Lewis acids or non-oxidizing acids known in the art. Other solvents can also be used, for example, benzene, toluene, chloroform, dichloromethane, etc.

In other embodiments, anhydrous MgSO₄ or another suitable agents such as Na₂SO₄, CaSO₄, and CaCl₂), known in the art is used in place of the MgSO₄.

A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. 

What is claimed is:
 1. A method for catalytic modification of cannabidiol (CBD) into a resulting final reaction product composition comprised substantially of Δ⁹-THC and Δ⁸-THC, the method comprising the steps of: providing a first extract, aliquot or sample of CBD; mixing the CBD with a first reaction solvent; adding a catalyst to the CBD and the first reaction solvent to generate a first reaction mixture containing primarily CBD; reacting the CBD with the catalyst and the first reaction solvent, under reaction conditions of about 0° C., to prepare a modified portion and an unmodified CBD portion of the reaction mixture together comprising CBD, Δ9-THC and Δ8-THC; adding a neutralizing agent to the first reaction mixture when about 70% of the CBD remains and about 30% of the CBD in the first reaction mixture has been modified to Δ9-THC and Δ8-THC creating the modified portion; removing substantially all of the catalyst and the neutralizing agent from the first reaction mixture to generate a first reaction product comprising CBD, Δ9-THC and Δ8-THC, wherein the modified portion of the first reaction product is 75% or more Δ9-THC and Δ8-THC; removing the first reaction solvent; dissolving the first reaction product in elution solution; loading the dissolved first reaction product onto a first chromatography column; eluting the dissolved first reaction product from the first column with an or at least one elution solution; collecting, in a first elution fraction, the CBD in the first reaction product from the elution solution; collecting, in a second elution fraction, the Δ9-THC and Δ8-THC in the first reaction product from the elution solution; removing all or substantially most of the elution solvent from the first and second elution fractions, thereby obtaining a final reaction product composition comprising Δ9-THC and Δ8-THC; and discarding or recycling through the first reaction mixture any fraction of the elution which is not THC, wherein the catalyst includes p-toluenesulfonic acid and the catalyst is free of boron trifluoride and free of boron trifluoride diethyl etherate.
 2. The method of claim 1 wherein the catalyst is added before, simultaneous with or after addition of the first reaction solvent to the CBD.
 3. The method of claim 1 wherein the reaction conditions comprise a time of between about 1 to 30 hours.
 4. The method of claim 1 wherein the removing the catalyst step further comprises adding a neutralizing agent and water whereby the neutralizing agent and water in the reaction mixture create a salt of the catalyst that reports to a water phase and is decanted off.
 5. The method of claim 1 wherein the first chromatography column is a normal phase chromatography column or a reverse phase chromatography column.
 6. The method of claim 1 wherein eluting from the column comprises use of a gradient or isocratic elution process.
 7. The method of claim 1 wherein in the first chromatography column the Δ9-THC and Δ8-THC are reversibly bound.
 8. The method of claim 3, wherein the reaction conditions comprise a time of about 4 to 24 hours.
 9. The method of claim 3, wherein the reaction conditions comprise a time of about 6 to 15 hours.
 10. The method of claim 3, wherein the reaction conditions comprise a time of about 8 hours. 